Saccharide-containing protein conjugates and uses thereof

ABSTRACT

Conjugates of a saccharide and a biomolecule, covalently linked therebetween via a non-hydrophobic linker and methods of preparing same are disclosed. Also disclosed are medical uses utilizing such conjugates. Glycosylation reagents for use in preparing these conjugates are also disclosed. Glycosylated proteins, characterized by improved performance, are also disclosed.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/673,987 filed on Feb. 18, 2010, which is a National Phase of PCTPatent Application No. PCT/IL2008/001143 having International filingdate of Aug. 20, 2008, which claims the benefit of priority of U.S.Provisional Patent Application No. 60/935,587 filed on Aug. 20, 2007.The contents of the above applications are all incorporated herein byreference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates tosaccharide-containing active reagents (e.g., glycosylation agents), toconjugates thereof with biomolecules and to methods utilizing theseconjugates in e.g., therapeutic applications. More particularly, but notexclusively, the present invention, in some embodiments thereof, relatesto saccharide-containing active reagents (e.g., glycosylation agents),to protein conjugates made therefrom and to methods utilizing theseprotein conjugates in therapeutic applications.

The trafficking of many proteins, and especially lysosomal enzymes, totheir target organs, cells and organelles is controlled and enabled bydifferent carbohydrate-specific receptors, such as the mannose ormannose-6-phosphate (M6P) receptors.

In order for proteins to be recognized and transported bycarbohydrate-specific receptors, the proteins should be glycosylatedwith oligosaccharide residues terminating with the appropriatecarbohydrate moiety. This is especially important in therapeuticproteins which should be transported to their action site in order toexert their therapeutic benefits.

Of particular importance are the mannose-6-phosphate receptors. Thebiogenesis of lysosomes, which are key components of the degradativemachinery of eukaryotic cells, requires the action of themannose-6-phosphate receptors (MPRs). Two MPRs, the 300-kDacation-independent MPR (CI-MPR) and the 46-kDa cation-dependent MPR(CD-MPR), participate in the intracellular delivery of 50 differentlysosomal enzymes to the lysosome by diverting these soluble acidhydrolases from the secretory pathway and delivering them from the transGolgi network to endosomal compartments. The CI-MPR has also been shownto function in the binding and internalization of ligands at the cellsurface. The higher capacity of the CI-MPR as compared with the CD-MPRin sorting lysosomal enzymes to the lysosome is due in part to theability of the CI-MPR at the cell surface to re-capture lysosomalenzymes that may have been secreted, resulting in their internalizationand delivery to endosomal compartments.

In addition to lysosomal enzymes, the repertoire of extracellularM6P-containing ligands has expanded in recent years to include a diversespectrum of proteins including the precursor form of transforming growthfactor and renin, proliferin, granzymes A and B, CD26, and herpessimplex viral glycoprotein D. Several studies have implicated theinteraction of these ligands with the CI-MPR at the cell surface asbeing essential to their activity and/or function, thus expanding therole of the CI-MPR from solely an intracellular protein carrier to acell surface signaling molecule via its lectin activity.

Oligosaccharides containing M6P have been isolated from lysosomal enzymemixtures from human skin fibroblasts, mouse lymphoma cells, murinemacrophages, and from purified lysosomal enzymes includingβ-glucuronidase and β-galactosidase. The results of structural analysesindicated that α(1,2)-linked M6P residues are present as terminal orpenultimate residues on either, or both, antennary arms of N-linkedoligomannosides. In addition, some M6P residues containN-acetylglucosamine in phosphodiester linkage. The binding properties ofthe oligomannosides obtained from acid hydrolases have been examined byaffinity chromatography on immobilized CI-MPR and CD-MPR. Theimmobilized receptors bind oligomannosides containing two M6P residueswith a greater affinity than those containing a single M6P residue.

As mentioned above, the glycosylation profile of a protein determinesits bioavailability and trafficking behavior. The glycosylation profileof a specific protein is highly dependent on its biosynthetic pathwayand its expressing platform. Thus, recombinant therapeutic proteins,expressed in various expressing platforms, including bacteria, fungi,plants, and mammalian cells, almost always differ dramatically in theirglycosylation sites, glycosylation level and oligosaccharide profilefrom the original human protein. These differences dramatically diminishthe bioavailability and uptake of the recombinant protein by the targetcells, resulting in diminished therapeutic potency or necessitatinghigher doses.

For example, recombinant lysosomal enzymes obtained from differentexpression systems are often not sufficiently phosphorylated and thelevel of M6P in the oligosaccharides varies considerably from oneexpression system to another. The high uptake form of α-galactosidase Ais bis-phosphorylated while only 20% of the a-galactosidase A expressedin CHO cells are phosphorylated and only 5% are bis-phosphorylated.Moreover, recombinant proteins expressed in plants, insect cells oryeasts do not have any M6P phosphorylation since these expressionsystems lack the M6P targeting pathway.

In view of the need to enable the targeting of recombinant therapeuticproteins, and especially of plant recombinant proteins, tocarbohydrate-specific receptors, such as the M6P receptor, a fewmethodologies have been devised to conjugate M6P to proteins, asfollows.

U.S. Pat. No. 7,001,994 teaches oxidizing protein glycosides tocarbonyls (aldehydes) and reacting these carbonyls with phosphorylatedmannopyranosyl oligosaccharides. The phosphorylated mannopyranosylsaccharides are derivatized with a carbonyl reactive substituent, suchas hydrazine, which upon reaction with the aldehydes yields a covalenthydrazone bond. This methodology was also exemplified by Cheng et al.(Abstracts of Papers, 232nd ACS National Meeting, San Francisco, Calif.,United States, Sep. 10-14, 2006) by attaching a syntheticoligosaccharide ligand bearing M6P residues in the optimal configurationfor binding the CI-MPR. In this methodology, the protein undergoesoxidative conditions which are not selective and may cause oxidativedamage to additional sites on the protein, including active sites. Suchoxidative damage may also promote oxidative stress upon administration.

Beljaars et al. (Liver 2001, 21, 320-328) have synthesized M6P-modifiedalbumin (M6P₂₈-HSA) in order to improve targeting of drugs to hepaticstellate cells. In this publication, it has been shown that the bindingof M6P-HSA to the M6P/IGFII receptor is specific. Furthermore, M6P₂₈-HSAwas extensively internalized by these cells. Using monensin, a specificinhibitor of the lysosomal pathway, proof was obtained that M6P-HSA isendocytosed via this route. Beljaars et al. have concluded thatM6P₂₈-HSA is applicable as a stellate cell-selective carrier forantifibrotic drugs that act intracellularly. The M6P was connected tothe albumin protein by phosphorylatingp-nitrophenyl-α-D-mannopyranoside, and further reducing the nitro-groupto the primary amine. The latter could be coupled to proteins by twomethodologies: In the first methodology the p-aminophenyl-sugar wasreacted with sodium nitrite under acidic conditions to form thediazonium salt of the derivatized sugar. This diazonium salt readilybinds covalently to tyrosine or histidyl residues on the protein. Inanother optional methodology the p-aminophenyl-sugar is treated withthiophosgen to convert the primary amine to an isothiocyanate group, thelatter readily reacts with primary amino groups on the protein,primarily lysine residues.

In a different methodology (see, U.S. patent application Ser. No.10/024,197) a phosphorylated glucocerebrosidase has been prepared whileutilizing isolated GlcNAc phosphotransferase and a phosphodiesterα-GlcNAcase. According to this methodology, which mimics naturalbiosynthetic mechanisms, both enzymes used in the phosphorylationprotocol should be produced and isolated, rendering this methodologycumbersome, cost-ineffective and laborious, necessitating thepurification of the therapeutic protein from the additional enzymes usedin its post-translational modification.

Lee et al. (Glycoconjugate Journal 2006, 23(5/6), 317-327) haveconjugated galactose-6-phosphate (Gal6P) to bovine serum albumin byusing glycosides of Gal6P that can potentially generate a terminalaldehyde group, namely Gal6P with a glycerol attached to its anomericcarbon. ω-Aldehydro glycosides were then conjugated to BSA via reductiveamination.

Hydrocarbon chains have been used as linkers to oligosaccharides indifferent biological studies. In WO 92/22662, oligosaccharides havingattached thereto an 8-methoxycarbonyloctyl [—(CH₂)₈CO₂CH₃] group, anduse thereof in a variety of biological applications are taught. Thispublication also teaches that a PEG chain can be used as the linker.

Distler et al. (J. Biol. Chem. 1991, 266, 21687) have also used an8-methoxycarbonyloctyl alkyl linker to show the binding specificity ofdifferent phosphorylated mannose glycosides to MPR. A series ofchemically synthesized oligomannosides that contain M6P residues wereutilized as inhibitors of the binding of β-galactosidase to CI-MPR andCD-MPR in order to probe the specificity of each receptor.

Tomoda et al. (Carbohydrate Research 1991, 213, 37-46) have also studiedthe binding of bovine serum albumin (BSA) derivatized withpenta-D-mannose-6-phosphate and have established that the best bindingwas obtained when the linkage mode between the terminal M6P sugar groupand the penultimate sugar residue was α(1→2). Furthermore, they haveshown that the length of the sugar chain also affects the binding to theM6P receptor, such that, for example, trisaccharides containing aterminal M6P group were more potent inhibitors than disaccharides.

JP 04210221 describes long chain alkyl D-glucoside-6-phosphates assurfactants for dishwashing and shampoos.

Cowden et al. (U.S. Pat. No. 6,294,521) describe the preparation ofsugar phosphates as anti-inflammatory agents. D-mannoside-6-phosphatederivatives wherein the anomeric carbon is derivatized with a long chainhydrocarbon (C₈-C₁₆) are used in treating inflammatory diseases,particularly cell-mediated inflammatory diseases.

Similar glucose-6-phosphate derivatives have been described by Jones etal. (Journal of the Chemical Society, Chemical Communications 1994, 11,1311-12). Dodecyl β-D-glucopyranoside-6-phosphate was used as a novelsurfactant possessing a long-chain hydrocarbon tail and a hydrophilichead, consisting of a phosphoryl group covalently linked to a(homochiral) glucose moiety. This compound has been successfully used inmiscellar electrokinetic capillary chromatography.

Short PEG chains (n=3) attached at one end to the anomeric carbon of amonosaccharide in a β-conformation and to a fatty acid at the other endare used as detergents in various pharmaceutical and cosmeticapplications (see, for example, JP 2766141, JP 2854203, JP 06080686, WO2006/098415). These publications suggest that such detergents (forexample, saccharide-(OCH₂CH₂)₃—NH—CO—CH(C₁₆H₃₃)₂) can be used in thepreparation of drug delivery systems, mainly liposomes, in combinationwith other lipids, such as cholesterol and glycerophospholipids.

Similar detergents, which were further used as anchored cryo-protectors,have been prepared by Wilhelm et al. (Liebigs Annalen 1995, 9, 1673-9),using long-chain alcohols (C₁₆) with 0-4 ethoxy spacers. Engel et al.(Journal of Pharmaceutical Sciences 2003, 92(11), 2229-2235) have usedthe same detergents in the creation of liposomes and have evaluated theinteraction of mannose and glucose derivatives of these detergents withConcanavalin A lectin. Engel et al. (Pharmaceutical Research 2003,20(1), 51-57) have also improved the uptake of such mannosyl-basedliposomes by macrophages, by enhancing the affinity towards mannosereceptor. The researchers have suggested that such mannosides withsufficiently long spacer arms are of potential use in receptor-mediatedtargeting of liposomes made with such detergents.

Millqvist-Fureby et al. have described (Biotechnology and Bioengineering1998, 59(6), 747-753) the synthesis of ethoxylated glycosides(tetraethylene glycol β-D-glucoside, tetraethylene glycol β-D-xyloside,and methoxy triethyleneglycol β-D-glucoside). These were in turn used asraw materials for the preparation of the above discussed detergents,bearing an additional fatty acid, such as ω-O-oleoyl tetraethyleneglycol β-D-glucoside.

These glycoside-PEG-fatty acid detergents were widely investigated fortheir physical properties (Czichocki et al. Journal of Chromatography, A2002, 943(2), 241-250; Zimmermann et al. Spectroscopy of BiologicalMolecules: New Directions, European Conference on the Spectroscopy ofBiological Molecules, 8th, Enschede, Netherlands, Aug. 29-Sep. 2, 1999(1999), 353-354) as well as in various applications, such as biofilminhibitory and removal agents (JP 2006347941) and for anticariesdentifrices (WO 2006/035821).

PEGylated glycosides, in which the glycosides are conjugated to a smallnumber of ethylene glycol groups, are commercially available althoughnot wide-spread. These glycoside-PEG derivatives are composed ofshort-chain PEGs (n≦4) and are mainly used to create the surfactantsdescribed above by their further conjugation to a hydrophobic moiety,such as a fatty acid.

WO 2005/093422 describes the use of bio-functionalized quantum dotscomprising a saccharide derivatized at the anomeric carbon in aβ-conformation with a PEG chain (n=6) derivatized at the other end withan alkyl thiol group. These bio-functionalized quantum dots can be usedin biological and medical research, imaging and/or therapy applications.

JP 3001381 discloses the use of monosaccharides linked to a PEG chain oran alkyl chain, and further linked to a polysaccharide, such aschitosan, a pullulan, a dextran, mannoglucan, heparin or hyaluronicacid. These moieties were used for delivering drugs following theirphysical incorporation in the matrices of these polysaccharides,similarly to liposomes.

Andersson et al. (Glycoconjugate Journal 1993, 10(3), 197-201) haveprepared glycosides with a linker of a short PEG chain (n=2 or 4)attached to the anomeric carbon (β-conformation). These glycosides wereconjugated to proteins through a terminal activated ester. The targetapplication of such glycoside-protein conjugates is as antigens indiagnostic tests or as immunogens. While conjugation withmonosaccharides is described, these studies were mainly practiced witholigosaccharides. The PEG linker in all the practiced conjugates wasconnected to the glycoside via an equatorial bond (β conformation). Asnoted above, the PEG chains used in this work were relatively short (n=2or 4).

Biskup et al. (ChemBioChem 2005, 6(6), 1007-1015) have used glycosidewith short PEG spacers (n=4) for immobilization of the glycoside ontosolid surfaces, followed by interacting the immobilized glycosides withlectins. It appears that the PEG spacers in the studied conjugates wereattached to the glycoside anomeric carbon in the β conformation.

Zalipsky et al. (Chemical Communications (Cambridge) 1999, 7, 653-654)have presented a practical approach for preparinggalactose-PEG-distearoylphosphatidic acid (DSPA) that retains fulllectin binding. Their methodology involved glycosylation of monobenzylether-PEG, suitable protection of the sugar hydroxy groups, anddebenzylation, followed by enzymatic transphosphatidylation withphosphatidylcholine and final deprotection.

WO 2006/093524 describes compositions comprising antigen-carbohydrateconjugates and methods of immune modulation utilizing these reagents.Thus, ovalbumin, a model antigen, was reacted with theN-hydroxy-succinimide (NHS) group of a bi-functional short hydrocarbonlinker to introduce maleimide functional groups. The latter were thenreacted with thiol-terminated short PEG chains (n=2-3) attached tosaccharides. These saccharides comprise monosaccharides, but mainlyhigh-mannose oligosaccharide. These conjugates were shown to enhanceantigen uptake and presentation to T cells, when compared to unmodifiedovalbumin. This, in turn, led to improved antigen-specific T cellactivation. The bond conformation between the short thiol-terminated PEGchains and the anomeric carbon was both α and β.

SUMMARY OF THE INVENTION

The prior art teaches conjugates of saccharides and proteins in whicheither a terminal saccharide in an oligosaccharide chain or a saccharidehaving attached thereto an alkyl chain were utilized. Some studies teachsaccharides having attached thereto a PEG chain, whereby the PEG chainserves as a linker to attach mainly agents such as fatty acids but insome cases proteins. Nonetheless, in these studies, the PEG chain thatwas utilized was relatively short (mostly up to 4 and no more than 6ethylene glycol units) and furthermore, the PEG was attached to theanomeric carbon via an equatorial bond (β conformation).

While conceiving the present invention, it was envisioned that proteinsconjugated to monosaccharides via a medium-to-long hydrophobic linkerchain (e.g., a PEG-containing linker having more than 6 alkylene glycolunits) would exhibit an improved performance.

It was further envisioned that such conjugates would exhibit an improvedperformance if the bond conformation between the saccharide moiety andthe linker would mimic the conformation between the terminal andpenultimate saccharides of naturally occurring protein glycosides. Thus,for example, it was envisioned that conjugates in which the saccharidemoiety is M6P or sialic acid, having an ether bond in an α conformationbetween the anomeric carbon of the saccharide and the linker, wouldexhibit an improved performance.

According to an aspect of some embodiments of the present inventionthere is provided a conjugate comprising a biomolecule and a saccharidemoiety being covalently linked thereto via a non-hydrophobic linker.

According to some embodiments of the invention, the linker is anon-saccharide moiety.

According to some embodiments of the invention, the linker is attachedto an anomeric carbon of the saccharide moiety.

According to some embodiments of the invention, the linker is attachedto the anomeric carbon via a bond having an α configuration.

According to an aspect of some embodiments of the present inventionthere is provided a conjugate comprising a biomolecule and a saccharidemoiety being covalently linked thereto via a non-hydrophobic linker,wherein the linker is attached to an anomeric carbon of the saccharidemoiety.

According to an aspect of some embodiments of the present inventionthere is provided a conjugate comprising a biomolecule and a saccharidemoiety being covalently linked thereto via a non-hydrophobic linker,wherein the linker is attached to an anomeric carbon of the saccharidemoiety via a bond having α configuration.

According to some embodiments of the invention, the linker comprises apoly(alkylene glycol) chain of at least 18 atoms in length.

According to an aspect of some embodiments of the present inventionthere is provided a conjugate comprising a biomolecule and a saccharidemoiety being covalently linked thereto via a poly(alkylene glycol)linker of at least 18 atoms in length.

According to an aspect of some embodiments of the present inventionthere is provided a conjugate comprising a biomolecule and a saccharidemoiety being covalently linked thereto via a poly(alkylene glycol)linker of at least 18 atoms in length, wherein the linker is attached toan anomeric carbon of the saccharide moiety via a bond having αconfiguration.

According to some embodiments of the invention, the poly(alkyleneglycol) comprises poly(ethylene glycol) (PEG).

According to some embodiments of the invention, the poly(alkyleneglycol) is from 24 to 36 atoms in length.

According to some embodiments of the invention, the poly(ethyleneglycol) comprises from 8 to 12 ethylene glycol units.

According to some embodiments of the invention, the linker comprises atleast two chemical moieties which are covalently linked to one another.

According to some embodiments of the invention, the linker comprises atleast two poly(alkylene glycol) moieties which are covalently linked toone another.

According to some embodiments of the invention, the at least twochemical moieties are the same or different.

According to some embodiments of the invention, the at least twochemical moieties form a linear linker.

According to some embodiments of the invention, the at least twochemical moieties form a branched linker.

According to some embodiments of the invention, the saccharide moiety isa monosaccharide.

According to some embodiments of the invention, the monosaccharide is asialic acid.

According to some embodiments of the invention, the conjugate has theformula:

wherein B is the biomolecule.

According to some embodiments of the invention, the biomolecule isattached via a thiol group in the biomolecule.

According to some embodiments of the invention, the biomolecule is aprotein and the thiol forms a part of a cysteine residue in the protein.

According to some embodiments of the invention, the biomolecule is aprotein and the thiol forms a part of a thiolated lysine residue in theprotein.

According to some embodiments of the invention, the conjugate has theformula:

wherein B is the biomolecule.

According to some embodiments of the invention, the biomolecule isattached via an amine group in the biomolecule.

According to some embodiments of the invention, the biomolecule is aprotein and the amine forms a part of a lysine residue of the protein.

According to some embodiments of the invention, the monosaccharide is ahexose.

According to some embodiments of the invention, the hexose is aD-hexose.

According to some embodiments of the invention, the linker is attachedto the anomeric carbon via a bond having α configuration.

According to some embodiments of the invention, the monosaccharide isselected from the group consisting of a mannose and a M6P.

According to some embodiments of the invention, the conjugate has theformula:

wherein B is the biomolecule.

According to some embodiments of the invention, the conjugate has theformula:

wherein B is the biomolecule.

According to some embodiments of the invention, the conjugate has theformula:

wherein B is the biomolecule.

According to some embodiments of the invention, the conjugate has theformula:

wherein B is the biomolecule.

According to some embodiments of the invention, the biomolecule isattached to the linker via an amine group in the biomolecule.

According to some embodiments of the invention, the biomolecule is aprotein and the amine forms a part of a lysine residue of the protein.

According to some embodiments of the invention, the conjugate has theformula:

wherein B is the biomolecule.

According to some embodiments of the invention, the conjugate has theformula:

wherein B is the biomolecule.

According to some embodiments of the invention, the conjugate has theformula:

wherein B is the biomolecule.

According to some embodiments of the invention, the biomolecule isattached via a thiol group in the biomolecule.

According to some embodiments of the invention, the biomolecule is aprotein and the thiol forms a part of a cysteine residue in the protein.

According to some embodiments of the invention, the biomolecule is aprotein and the thiol forms a part of a thiolated lysine residue in theprotein.

According to some embodiments of the invention, the biomolecule isselected from the group consisting of a protein, a peptide, anoligonucleotide, an antisense, a polynucleotide, a hormone, a steroid,an antibody, an antigen, a toxin, a growth factor, an agonist, anantagonist, a co-factor, a cytokine residue, an enzyme, animmunoglobulin, an inhibitor, a ligand, a prostaglandin, a vaccine and avitamin.

According to some embodiments of the invention, the biomolecule is aprotein.

According to some embodiments of the invention, the protein is arecombinant protein produced by a host cell.

According to some embodiments of the invention, the protein is plantcell produced recombinant protein.

According to some embodiments of the invention, the biomolecule isselected from the group consisting of a lysosomal protective protein,L-iduronidase, iduronate-2-sulfatase, heparan-N-sulfatase,α-N-acetylglucosaminidase, acetylCoA:α-glucosaminide acetyltransferase,N-acetylglucosamine-6-sulfatase, galactose-6-sulfatase, β-galactosidase,N-acetylgalactosamine-4-sulfatase, β-glucuronidase,hyaluronoglucosaminidase, aspartylglucosaminidase, acid lipase, cystinetransporter, Lamp-2, α-galactosidase A, acid ceramidase, α-L-fucosidase,glucocerebrosidase, galactocerebrosidase, α-glucosidase,β-galactosidase, β-hexosaminidase A, β-hexosaminidase B, ganglioside GM2activator protein, α-D-mannosidase, β-D-mannosidase, arylsulfatase A,saposin B, neuraminidase, phosphotransferase, palmitoyl proteinthioesterase, tripeptidyl peptidase I, acid sphingomyelinase, cathepsinK, α-galactosidase B, sialic acid transporter, tartrate-resistant acidphosphatase, asparaginase, ceroid lipofuscinosis neuronal protein 5,CPVL, cathepsin B, dipeptidyl-peptidase I, cathepsin D, cathepsin H,cathepsin L, cathepsin S, cathepsin Z, deoxyribonuclease II,dipeptidyl-peptidase II, N-acetylgalactosamine-6-sulfatase, γ-glutamylhydrolase, heparanase, legumain, 1-O-acylceramide synthase,myeloperoxidase, α-N-acetylgalactosaminidase, NPC2 protein, plasmaglutamate carboxypeptidase, Pro-X carboxypeptidase, proactivatorpolypeptide, N-sulfoglucosamine sulfohydrolase, sialic acid9-O-acetylesterase, tripeptidyl-peptidase I, lactotransferrin,pancreatic ribonuclease, hornerin, cation-dependent mannose-6-phosphatereceptor, ribonuclease K6, intercellular adhesion molecule 1, CREG1protein, laminin A, hemoglobin ζ chain, cerebellin 4, desmoplakin, fattyacid-binding protein, sulfatase-modifying factor, leukocyte elastase,procollagen-lysine-2-oxoglutarate-5-dioxygenase 1, ferritin light chain,acid sphingomyelinase-like phosphodiesterase 3A, hemoglobin β chain,ribonuclease T2, cat eye syndrome critical region 1, leucine-richα₂-glycoprotein, antithrombin-III, serum amyloid P-component, plasmaserine protease inhibitor, haptoglobin-related protein, complement C1qsubcomponent A chain, complement C1q subcomponent B chain, complementC1q subcomponent C chain, cholinesterase, angiotensinogen,prostaglandin-H₂ D-isomerase, plasma protease Cl inhibitor, mammalianependymin-related protein, α₁B-glycoprotein, plasma kallikrein,hemopexin, AMBP protein, α₁-antitrypsin, pigment epithelium-derivedfactor, α₂-macroglobulin, kallistatin, Fc fragment of IgG-bindingprotein, corticosteroid-binding globulin, zinc-α₂-glycoprotein, afamin,serotransferrin, ceruplasmin, biotimidase, ficolin-3, serum albumin,α₁-acid glycoprotein 1, α₁-acid glycoprotein 2, CD5 antigen-like,complement C2 precursor, complement C3 precursor, inter-α-trypsininhibitor heavy chain H4, inter-α-trypsin inhibitor heavy chain 2,inter-α-trypsin inhibitor heavy chain 1, ficolin-2, complement factor B,dopamine β-hydroxylase, fibrinogen β chain, α₁-antichymotrypsin,extracellular matrix protein 1, kininogen-1, lumican, complementcomponent 4B, cation-independent mannose-6-phosphate receptor,adipocyte-derived leucine aminopeptidase, fetuin-B,N-acetylmuramoyl-L-alanine amidase, histidine-rich glycoprotein,vitronectin, α₂-HS-glycoprotein, clusterin, C4b-binding protein α chain,mannan-binding lectin serine protease 1, and transthyretin.

According to some embodiments of the invention, the protein is a greenfluorescent protein.

According to some embodiments of the invention, the biomolecule is alysosomal protein.

According to some embodiments of the invention, the lysosomal protein isa glucocerebrosidase.

According to some embodiments of the invention, the lysosomal protein isan α-galactosidase.

According to some embodiments of the invention, the monosaccharide isM6P.

According to some embodiments of the invention, the monosaccharide is asialic acid.

According to some embodiments of the invention, the sialic acid isN-acetylneuraminic acid.

According to some embodiments of the invention, the biomolecule is afollicle-stimulating hormone (FSH).

According to some embodiments of the invention, the linker is attachedto the biomolecule via a covalent bond formed between a reactive groupin the linker and a functional group on the biomolecule.

According to some embodiments of the invention, the reactive group is acarboxylate and the functional group is an amine.

According to some embodiments of the invention, the biomolecule is aprotein and the amine forms a part of a lysine residue of the protein.

According to some embodiments of the invention, the reactive group is anamine and the functional group is a carboxylate.

According to some embodiments of the invention, the biomolecule is aprotein and the carboxylate forms a part of a glutamate residue and/oran aspartate residue in the protein.

According to some embodiments of the invention, the reactive group is amaleimide and the functional group is a thiol.

According to some embodiments of the invention, the biomolecule is aprotein and the thiol forms a part of a cysteine residue in the protein.

According to some embodiments of the invention, the biomolecule is aprotein and the thiol forms a part of a thiolated lysine residue in theprotein.

According to some embodiments of the invention, an uptake of theconjugate into cells is at least 10% higher than an uptake of thebiomolecule into the cells.

According to an aspect of some embodiments of the present inventionthere is provided a conjugate having the formula:

wherein:

n=8;

W is selected from the group consisting of —CH₂CH₂C(═O)— and—CH₂CH₂—NHCOCH₂CH₂-maleimide; and

B is an α-galactosidase.

According to an aspect of some embodiments of the present inventionthere is provided a conjugate having the formula:

wherein:

n=8;

W is selected from the group consisting of —CH₂CH₂C(═O)— and—CH₂CH₂—NHCOCH₂CH₂-maleimide; and

B is a glucocerebrosidase.

According to an aspect of some embodiments of the present inventionthere is provided a conjugate having the formula:

wherein:

n=8;

W is selected from the group consisting of —CH₂CH₂C(═O)— and—CH₂CH₂—NHCOCH₂CH₂-maleimide; and

B is a green fluorescent protein.

According to an aspect of some embodiments of the present inventionthere is provided a process of preparing a conjugate described herein,the process comprising reacting a glycosylation reagent, which comprisesthe saccharide moiety having attached thereto a non-hydrophobic linkerhaving a reactive group, with the biomolecule.

According to some embodiments of the invention, the reactive group isselected from the group consisting of an amine, a maleimide and acarboxylate.

According to some embodiments of the invention, the reactive group is acarboxylate, and the carboxylate is reacted with an amine-containingmoiety on the biomolecule.

According to some embodiments of the invention, the biomolecule is aprotein and the amine forms a part of a lysine residue of the protein.

According to some embodiments of the invention, the reactive group is anamine, and the amine is reacted with a carboxylate on the biomolecule.

According to some embodiments of the invention, the biomolecule is aprotein and the carboxylate forms a part of a glutamate and/or anaspartate residue in the protein.

According to some embodiments of the invention, the reactive group is amaleimide, and the maleimide is reacted with a thiol on the biomolecule.

According to some embodiments of the invention, the biomolecule is aprotein and the thiol forms a part of a cysteine residue in the protein.

According to some embodiments of the invention, the biomolecule is aprotein and the thiol forms a part of a thiolated lysine residue in theprotein.

According to some embodiments of the invention, the glycosylationreagent is selected from the group consisting of:

According to some embodiments of the invention, the glycosylationreagent has the formula:

wherein:

n=8; and

Z is selected from the group consisting of —CH₂CH₂CO₂H and—CH₂CH₂—NHCOCH₂CH₂-maleimide.

According to some embodiments of the invention, the biomolecule is anα-galactosidase.

According to some embodiments of the invention, the biomolecule is aglucocerebrosidase.

According to some embodiments of the invention, the biomolecule is agreen fluorescent protein.

According to some embodiments of the invention, the glycosylating agentand the biomolecule are reacted using a stoichiometric ratio of theglycosylation reagent to the biomolecule, the ratio being selected so asto control the average number of saccharide moieties conjugated to thebiomolecule.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the conjugatedescribed herein and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a use of a conjugate described herein as a fluorescentlabeling agent.

According to an aspect of some embodiments of the present inventionthere is provided a use of a conjugate described herein, in themanufacture of a medicament.

According to some embodiments of the invention, the protein is alysosomal protein and the medicament is for treating a metabolicdisease.

According to some embodiments of the invention, the disease is alysosomal storage disease.

According to some embodiments of the invention, the disease is selectedfrom the group consisting of mucopolysaccharidosis types I, II, IIIA,IIIB, IIIC, IIID, IVA, IVB, VI, VII and IX, aspartylglucosaminuria,cholesterol ester storage disease, cystinosis, Danon disease, Fabrydisease, Farber disease, fucosidosis, galactosialidosis, Gaucherdisease, globoid cell leucodystrophy, GM1-gangliosidosis, Tay Sachsdisease, Sandhoff disease, GM2-gangliosidosis, α-mannosidosis,β-mannosidosis, metachromatic leucodystrophy, mucolipidosis type I,mucolipidosis type II, mucolipidosis type IIIC, multiple sulfatasedeficiency, CLN1 Batten disease, CLN2 Batten disease, Niemann-Pickdisease types A, B and C, pycnodysostosis, Schindler disease and sialicacid storage disease.

According to some embodiments of the invention, the medicament is fortreating a protein-related disease.

According to some embodiments of the invention, the protein-relateddisease is associated with at least one protein selected from the groupconsisting of follicle-stimulating hormone, a lysosomal protectiveprotein, L-iduronidase, iduronate-2-sulfatase, heparan-N-sulfatase,α-N-acetylglucosaminidase, acetylCoA:α-glucosaminide acetyltransferase,N-acetylglucosamine-6-sulfatase, galactose-6-sulfatase, β-galactosidase,N-acetylgalactosamine-4-sulfatase, β-glucuronidase,hyaluronoglucosaminidase, aspartylglucosaminidase, acid lipase, cystinetransporter, Lamp-2, α-galactosidase A, acid ceramidase, α-L-fucosidase,glucocerebrosidase, galactocerebrosidase, α-glucosidase,β-galactosidase, β-hexosaminidase A, β-hexosaminidase B, ganglioside GM2activator protein, α-D-mannosidase, β-D-mannosidase, arylsulfatase A,saposin B, neuraminidase, phosphotransferase, palmitoyl proteinthioesterase, tripeptidyl peptidase I, acid sphingomyelinase, cathepsinK, α-galactosidase B, sialic acid transporter, tartrate-resistant acidphosphatase, asparaginase, ceroid lipofuscinosis neuronal protein 5,CPVL, cathepsin B, dipeptidyl-peptidase I, cathepsin D, cathepsin H,cathepsin L, cathepsin S, cathepsin Z, deoxyribonuclease II,dipeptidyl-peptidase II, N-acetylgalactosamine-6-sulfatase, γ-glutamylhydrolase, heparanase, legumain, 1-O-acylceramide synthase,myeloperoxidase, α-N-acetylgalactosaminidase, NPC2 protein, plasmaglutamate carboxypeptidase, Pro-X carboxypeptidase, proactivatorpolypeptide, N-sulfoglucosamine sulfohydrolase, sialic acid9-O-acetylesterase, tripeptidyl-peptidase I, lactotransferrin,pancreatic ribonuclease, hornerin, cation-dependent mannose-6-phosphatereceptor, ribonuclease K6, intercellular adhesion molecule 1, CREG1protein, laminin A, hemoglobin ζ chain, cerebellin 4, desmoplakin, fattyacid-binding protein, sulfatase-modifying factor, leukocyte elastase,procollagen-lysine-2-oxoglutarate-5-dioxygenase 1, ferritin light chain,acid sphingomyelinase-like phosphodiesterase 3A, hemoglobin β chain,ribonuclease T2, cat eye syndrome critical region 1, leucine-richα₂-glycoprotein, antithrombin-III, serum amyloid P-component, plasmaserine protease inhibitor, haptoglobin-related protein, complement C1qsubcomponent A chain, complement C1q subcomponent B chain, complementC1q subcomponent C chain, cholinesterase, angiotensinogen,prostaglandin-H₂ D-isomerase, plasma protease C1 inhibitor, mammalianependymin-related protein, α₁B-glycoprotein, plasma kallikrein,hemopexin, AMBP protein, α₁-antitrypsin, pigment epithelium-derivedfactor, α₂-macroglobulin, kallistatin, Fc fragment of IgG-bindingprotein, corticosteroid-binding globulin, zinc-α₂-glycoprotein, afamin,serotransferrin, ceruplasmin, biotimidase, ficolin-3, serum albumin,α₁-acid glycoprotein 1, α₁-acid glycoprotein 2, CD5 antigen-like,complement C2 precursor, complement C3 precursor, inter-α-trypsininhibitor heavy chain H4, inter-α-trypsin inhibitor heavy chain 2,inter-α-trypsin inhibitor heavy chain 1, ficolin-2, complement factor B,dopamine β-hydroxylase, fibrinogen 0 chain, α₁-antichymotrypsin,extracellular matrix protein 1, kininogen-1, lumican, complementcomponent 4B, cation-independent mannose-6-phosphate receptor,adipocyte-derived leucine aminopeptidase, fetuin-B,N-acetylmuramoyl-L-alanine amidase, histidine-rich glycoprotein,vitronectin, α₂-HS-glycoprotein, clusterin, C4b-binding protein α chain,mannan-binding lectin serine protease 1, and transthyretin.

According to some embodiments of the invention, the protein-relateddisease is associated with a deficiency of the at least one protein.

According to some embodiments of the invention, the conjugate localizesthe biomolecule in a tissue.

According to some embodiments of the invention, an activity of thebiomolecule in the conjugate is at least the same as an activity of anon-modified form of the biomolecule.

According to some embodiments of the invention, the medicament is usedin enzyme replacement therapy, hormone replacement therapy and/or as avaccine.

According to an aspect of some embodiments of the present inventionthere is provided a use of a conjugate having the formula:

wherein:

n=8;

W is selected from the group consisting of —CH₂CH₂C(═O)— and—CH₂CH₂—NHCOCH₂CH₂-maleimide-; and

B is an α-galactosidase,

in the manufacture of a medicament for treating Fabry disease.

According to an aspect of some embodiments of the present inventionthere is provided a use of a conjugate having the formula:

wherein:

n=8;

W is selected from the group consisting of —CH₂CH₂C(═O)— and—CH₂CH₂—NHCOCH₂CH₂-maleimide-; and

B is a glucocerebrosidase,

in the manufacture of a medicament for treating Gaucher's disease.

According to an aspect of some embodiments of the present inventionthere is provided a compound comprising a saccharide moiety and anon-hydrophobic linker being attached thereto.

According to some embodiments of the invention, the linker is anon-saccharide moiety.

According to some embodiments of the invention, the linker is attachedto an anomeric carbon of the saccharide moiety.

According to some embodiments of the invention, the linker is attachedto the anomeric carbon via a bond having an α configuration.

According to an aspect of some embodiments of the present inventionthere is provided a compound comprising a saccharide moiety and anon-hydrophobic linker being attached thereto, wherein the linker isattached to an anomeric carbon of the saccharide moiety.

According to an aspect of some embodiments of the present inventionthere is provided a compound comprising a saccharide moiety and anon-hydrophobic linker being attached thereto, wherein the linker isattached to an anomeric carbon of the saccharide moiety via a bondhaving α configuration.

According to some embodiments of the invention, the linker comprises apoly(alkylene glycol) chain of at least 18 atoms in length.

According to an aspect of some embodiments of the present inventionthere is provided a compound comprising a saccharide moiety and apoly(alkylene glycol) linker being attached thereto, the linker being ofat least 18 atoms in length.

According to an aspect of some embodiments of the present inventionthere is provided a compound comprising a saccharide moiety and apoly(alkylene glycol) linker being attached thereto, the linker being atleast 18 atoms in length, wherein the linker is attached to an anomericcarbon of the saccharide moiety via a bond having a configuration.

According to some embodiments of the invention, the poly(alkyleneglycol) comprises poly(ethylene glycol) (PEG).

According to some embodiments of the invention, the poly(alkyleneglycol) is from 24 to 36 atoms in length.

According to some embodiments of the invention, the poly(ethyleneglycol) comprises from 8 to 12 ethylene glycol units.

According to some embodiments of the invention, the linker comprises atleast two chemical moieties which are covalently linked to one another.

According to some embodiments of the invention, the linker comprises atleast two poly(alkylene glycol) moieties which are covalently linked toone another.

According to some embodiments of the invention, the at least twochemical moieties are the same or different.

According to some embodiments of the invention, the at least twochemical moieties form a linear linker.

According to some embodiments of the invention, the at least twochemical moieties form a branched linker.

According to some embodiments of the invention, the saccharide moiety isa monosaccharide.

According to some embodiments of the invention, the saccharide moiety isa hexose.

According to some embodiments of the invention, the monosaccharide isselected from the group consisting of a mannose and a M6P.

According to some embodiments of the invention, the linker comprises areactive group.

According to some embodiments of the invention, the linker terminates bythe reactive group.

According to some embodiments of the invention, the reactive group is ina protected form thereof.

According to some embodiments of the invention, the reactive group isselected from the group consisting of an amine, a maleimide and acarboxylate.

According to an aspect of some embodiments of the present inventionthere is provided a compound having the formula:

wherein:

n=8; and

Z is selected from the group consisting of —CH₂CH₂CO₂H and—CH₂CH₂—NHCOCH₂CH₂-maleimide.

According to an aspect of some embodiments of the present inventionthere is provided a compound selected from the group consisting of:

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

The term “comprising” means that other steps and ingredients that do notaffect the final result can be added. This term encompasses the terms“consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

As used herein, the singular form “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

As used herein the term “about” refers to ±10%.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. Some embodiments of the invention are hereindescribed, by way of example only, with reference to the accompanyingimages and drawings. With specific reference now to the images anddrawings in detail, it is stressed that the particulars shown are by wayof example and for purposes of illustrative discussion of embodiments ofthe invention. In this regard, the description taken with the imagesmakes apparent to those skilled in the art how embodiments of theinvention may be practiced.

In the drawings:

FIGS. 1A-B are depictions of exemplary linear glycosylation reagentscomprising M6P and either a carboxylic acid reactive group (FIG. 1A) ora maleimide reactive group (FIG. 1B);

FIGS. 2A-D are depictions of exemplary branched glycosylation reagentscomprising 3 M6P units (FIGS. 2A and 2C) or 2 M6P units (FIGS. 2B and2D);

FIGS. 3A-B are depictions of exemplary linear glycosylation reagentscomprising sialic acid and either a maleimide reactive group (FIG. 3A)or a carboxylic acid reactive group activated with N-hydroxysuccinimide(FIG. 3B);

FIG. 4 is a photograph of an SDS-PAGE gel showing molecular weightstandards (lanes 1 and 6), plant recombinant α-galactosidase-A (lanes 2and 4), plant recombinant α-galactosidase-A conjugated withM6P-PEG₈-CO₂H (lane 3) and plant recombinant α-galactosidase-Aconjugated with 750 Da mPEG-CO₂H;

FIG. 5 is a photograph of an isoelectric focusing (IEF) gel showing IEFpI standards (lanes 1 and 7), plant recombinant α-galactosidase-A (lanes2 and 4), plant recombinant α-galactosidase-A with dimethyl sulfoxide(lane 3), plant recombinant α-galactosidase-A conjugated withM6P-PEG₈-CO₂H (lane 5) and plant recombinant α-galactosidase-Aconjugated with 750 Da mPEG-CO₂H (lane 6);

FIG. 6 is a photograph of an IEF gel showing IEF pI standards (lanes 1and 5), plant recombinant α-galactosidase-A (lane 2), plant recombinantα-galactosidase-A conjugated with M6P-PEG₈-CO₂H (lane 3) and plantrecombinant α-galactosidase-A exposed to conjugation reaction conditionsin the absence of M6P-PEG₈-CO₂H (lane 4);

FIG. 7 is a photograph of an SDS-PAGE gel showing molecular weightstandards (left lanes 1), plant recombinant α-galactosidase-A (middlelane), and plant recombinant α-galactosidase-A conjugated withM6P-PEG₈-CO₂H (right lane);

FIGS. 8A-B are MALDI-TOF (Matrix-Assisted LaserDesorption/Ionization-Time Of Flight) mass spectra for plant recombinantα-galactosidase-A (FIG. 8A) and plant recombinant α-galactosidase-Aconjugated with M6P-PEG₈-CO₂H (FIG. 8B) showing a 5.1 kDa increase inthe mass of plant recombinant α-galactosidase-A upon conjugation withM6P-PEG₈-CO₂H;

FIG. 9 is a photograph of an isoelectric focusing (IEF) gel showing IEFpI standards (lane 1), plant recombinant glucocerebrosidase (GCD) (lane2), and plant recombinant GCD conjugated with M6P-PEG₈-CO₂H (lane 3);

FIG. 10 is a photograph of an SDS-PAGE gel showing plant recombinant GCD(lane 1), molecular weight standards (lane 2) and plant recombinant GCDconjugated with M6P-PEG₈-CO₂H (lane 3);

FIGS. 11A-B are MALDI-TOF (Matrix-Assisted LaserDesorption/Ionization-Time Of Flight) mass spectra for plant recombinantGCD (FIG. 8A) and plant recombinant GCD conjugated with M6P-PEG₈-CO₂H(FIG. 8B) showing a 2,488 Da increase in the mass of plant recombinantGCD upon conjugation with M6P-PEG₈-CO₂H;

FIGS. 12A-B are photographs of an SDS-PAGE gel (FIG. 12A) and anisoelectric focusing (IEF) gel (FIG. 12B) showing molecular weightstandards (FIG. 12A) or IEF pI standards (FIG. 12B) (lanes 1), GFP(green fluorescent protein) conjugated with M6P-PEG₈-CO₂H (lanes 2) andGFP (lanes 3);

FIGS. 13A-B are MALDI-TOF (Matrix-Assisted LaserDesorption/Ionization-Time Of Flight) mass spectra for GFP (FIG. 8A) andGFP conjugated with M6P-PEG₈-CO₂H (FIG. 8B) showing an increase in themass of GFP upon conjugation with M6P-PEG₈-CO₂H;

FIGS. 14A-B are photographs of an isoelectric focusing (IEF) gel (FIG.14A) and an SDS-PAGE gel (FIG. 14B) showing IEF pI standards (FIG. 14A,lanes 1 and 4) or molecular weight standards (FIG. 14B, lane 1), plantrecombinant α-galactosidase A (lanes 2) and plant recombinantα-galactosidase A thiolated with Traut's reagent and conjugated withM6P-PEG₈-maleimide (lanes 3);

FIGS. 15A-B are a photograph of a Western blot (FIG. 15A) and a bargraph (FIG. 15B) showing uptake of plant recombinant α-galactosidase-A,plant recombinant α-galactosidase-A conjugated with M6P-PEG₈-maleimide(FIG. 15A only), and plant recombinant α-galactosidase-A conjugated withM6P-PEG₁₂-CO₂H in Fabry fibroblasts;

FIGS. 16A-B are bar graphs presenting plant recombinantα-galactosidase-A activity in liver tissue samples of mice 24 hoursafter injection of 18.75 mg/kg plant recombinant α-galactosidase-A, or 3mg/kg or 10 mg/kg M6P-PEG₈-CONH-α-galactosidase-A as absoluteconcentrations (ng α-galactosidase-A per ml extract) (FIG. 16A) or asconcentrations normalized relative to 3 mg/kg of injectedα-galactosidase-A (FIG. 16B), wherein each bar represents mean resultsfrom tissues of 4 mice;

FIG. 17 is a photograph of a Western blot of α-galactosidase-A in mouseliver tissue samples collected 24 hours after injection of 18.75 mg/kgplant recombinant α-galactosidase-A, or 3 mg/kg (low) or 10 mg/kg (high)M6P-PEG₈-CONH-α-galactosidase-A, as well as of 25 ng of reference plantrecombinant α-galactosidase-A or MP6-PEG-α-galactosidase-A (M6PPEG₈ ref)in saline;

FIGS. 18A-B are bar graphs presenting plant recombinantα-galactosidase-A activity in spleen tissue samples of mice 24 hoursafter injection of 18.75 mg/kg plant recombinant α-galactosidase-A, or 3mg/kg or 10 mg/kg M6P-PEG₈-CONH-α-galactosidase-A as absoluteconcentrations (ng α-galactosidase-A per ml extract) (FIG. 18A) or asconcentrations normalized relative to 3 mg/kg of injectedα-galactosidase-A (FIG. 18B), wherein each bar represents mean resultsfrom tissues of 4 mice;

FIGS. 19A-B are bar graphs presenting α-galactosidase-A activity inheart tissue samples of mice 24 hours after injection of 18.75 mg/kgplant recombinant α-galactosidase-A, or 3 mg/kg or 10 mg/kgM6P-PEG₈-CONH-α-galactosidase-A as absolute concentrations (ngα-galactosidase-A per ml extract) (FIG. 19A) or as concentrationsnormalized relative to 3 mg/kg of injected α-galactosidase-A (FIG. 19B),wherein each bar represents mean results from tissues of 4 mice;

FIGS. 20A-B are bar graphs presenting α-galactosidase-A activity inliver tissue samples of mice 24 hours after injection of 18.75 mg/kgplant recombinant α-galactosidase-A, or 3 mg/kg or 10 mg/kgM6P-PEG₈-CONHα-galactosidase-A as absolute concentrations (ngα-galactosidase-A per ml extract) (FIG. 20A) or as concentrationsnormalized relative to 3 mg/kg of injected α-galactosidase-A (FIG. 20B),wherein each bar represents mean results from tissues of 4 mice;

FIGS. 21A-B are bar graphs presenting α-galactosidase-A activity inkidney tissue samples of mice 24 hours after injection of 18.75 mg/kgplant recombinant α-galactosidase-A, or 3 mg/kg or 10 mg/kgM6P-PEG₈-CONH-α-galactosidase-A as absolute concentrations (ngα-galactosidase-A per ml extract) (FIG. 21A) or as concentrationsnormalized relative to 3 mg/kg of injected α-galactosidase-A (FIG. 21B),wherein each bar represents mean results from tissues of 4 mice;

FIGS. 22A-B are bar graphs presenting α-galactosidase-A activity inspleen tissue samples of mice 7 days after injection of 18.75 mg/kgplant recombinant α-galactosidase-A, or 3 mg/kgM6P-PEG₈-CONH-α-galactosidase-A as absolute concentrations (ngα-galactosidase-A per ml extract) (FIG. 22A) or as concentrationsnormalized relative to 3 mg/kg of injected α-galactosidase-A (FIG. 22B),wherein each bar represents mean results from tissues of 4 mice;

FIG. 23 is a bar graph presenting α-galactosidase-A activity in livertissue samples of mice 7 days after injection of 18.75 mg/kg plantrecombinant α-galactosidase-A, or 3 mg/kgM6P-PEG₈-CONH-α-galactosidase-A as concentrations normalized relative to3 mg/kg of injected α-galactosidase-A;

FIG. 24 is a bar graph presenting α-galactosidase-A activity in hearttissue samples of mice 7 days after injection of 18.75 mg/kg plantrecombinant α-galactosidase-A, or 3 mg/kgM6P-PEG₈-CONH-α-galactosidase-A as concentrations normalized relative to3 mg/kg of injected α-galactosidase-A;

FIG. 25 shows photographs of Western blots of α-galactosidase-A inplasma samples 24 hours and 7 days after injection of 18.75 mg/kg plantrecombinant α-galactosidase-A, 3 mg/kg or 10 mg/kgM6P-PEG₈-CONH-α-galactosidase-A, or saline;

FIGS. 26A-B are bar graphs presenting α-galactosidase-A activity inheart (FIG. 26A) and lung (FIG. 26B) tissue samples of mice 24 hoursdays after injection of 3 mg/kg or 10 mg/kg of plant recombinantα-galactosidase-A, M6P-PEG₁₂-CONH-α-galactosidase-A orM6P-PEG₈-maleimide-α-galactosidase-A as concentrations normalizedrelative to 3 mg/kg of injected α-galactosidase-A;

FIGS. 27A-B are fluorescent microscopy photographs showing uptake of 1μM GFP (FIG. 27A) or M6P-PEG-CONH-GFP (FIG. 27B) into Fabry fibroblastsafter 24 hours of incubation; and

FIG. 28 is a photograph of an SDS-PAGE gel showing molecular weightstandards (MW), and plant recombinant α-galactosidase conjugated with100 molar equivalents of 5 kDa mPEG-COOH and either 100 (lanes 1, 4, 5and 8), 130 (lanes 2 and 6) or 200 (lanes 3 and 7) equivalents of eachof ECD and sulfo-N-hydroxysuccinimide.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention generally relates to glycosylation reagents whichcan be used in the preparation of modified biomolecules with improvedperformance and to modified biomolecules formed thereby. The modifiedbiomolecules of the present embodiments are conjugates composed of thebiomolecule and a saccharide moiety, being covalently linkedtherebetween via a linker, preferably being a non-hydrophobic,non-saccharide linker. The modification of the biomolecule (e.g., aprotein) is aimed at increasing the serum half-life of a biomolecule, orfacilitating the interaction of the resulting conjugate withcarbohydrate-specific receptors and thus enables the recognition andfurther trafficking of the modified biomolecule to target tissues, cellsor organelles and/or its uptake across cellular membranes viainteraction with the specific membranal receptor.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

The present inventors have designed and successfully prepared andpracticed a glycosylation reagent for forming a conjugate that comprisesa biomolecule (e.g., a protein), a saccharide moiety and a linkerlinking the biomolecule and the saccharide moiety.

As used herein, the phrase “glycosylation reagent” describes a compoundthat is capable of coupling (conjugating) a saccharide moiety to anothercompound, herein a biomolecule. This phrase is also referred to hereininterchangeably as a saccharide derivatization agent. When thesaccharide moiety is a mannose-6-phosphate (M6P), the phrase M6Pylationreagent is used interchangeably to described the glycosylation reagent.

The glycosylation reagents presented herein are based on a saccharidemoiety linked to a non-hydrophobic linker.

As used herein, the phrase “non-hydrophobic” describes a compound ormoiety that is substantially water-soluble. A detailed description ofthe linker moiety is provided hereinafter.

These reagents, referred to herein also as “compounds”, can becollectively represented by the following general Formula:

X-A-Z

wherein:

X is a saccharide moiety, such as, for example, a monosaccharide;

A is a non-hydrophobic linker, such as, for example, a poly(alkyleneglycol) chain at least 18 atoms in length; and

Z is a reactive group that forms a part of the linker.

As used herein, the phrase “reactive group” describes a chemical groupthat is capable of undergoing a chemical reaction that typically leadsto a bond formation. The bond, according to the present embodiments, ispreferably a covalent bond. Chemical reactions that lead to a bondformation include, for example, nucleophilic and electrophilicsubstitutions, nucleophilic and electrophilic addition reactions,alkylations, addition-elimination reactions, cycloaddition reactions,rearrangement reactions and any other known organic reactions thatinvolve a functional group, as well as combinations thereof.

The reactive group is selected suitable for undergoing a chemicalreaction that leads to a bond formation with a complementaryfunctionality in the biomolecule.

The reactive group may optionally comprise a non-reactive portion (e.g.,an alkyl) which may serve, for example, to attach a reactive portion ofthe reactive group to the A moiety.

The linker, as described herein, is represented by -A-Z in the aboveformula.

As used herein, the term “saccharide moiety” describes a moiety, asdefined herein, that contains one or more saccharide units.

As used herein the term “moiety” describes a major portion of a firstmolecule which is covalently linked to another molecule and whichretains its main structural features and/or activity. Thus, a “moiety”refers to a part of a molecule formed by conjugating the aforementionedfirst molecule to one or more other molecules, and represents thatportion of the first molecule that is present in the conjugationproduct. For example, a carboxylic acid moiety is the R—C(═O)— portionof a R—C(═O)OH carboxylic acid molecule formed upon conjugating thelatter to an amine group in a second molecule, to thereby obtain anamide. In another example, an alkyl moiety is the portion of an alkylhalide molecule formed upon a nucleophilic reaction between the alkylhalide and an electrophilic molecule.

Accordingly, a “saccharide moiety” is that portion of a saccharidemolecule formed upon conjugating a second molecule thereto.

In exemplary embodiments of the invention, the saccharide moietycontains one saccharide unit and the saccharide unit is amonosaccharide.

The term “monosaccharide”, as used herein and is well known in the art,refers to a simple form of a sugar that consists of a single saccharideunit which cannot be further decomposed to smaller saccharide buildingblocks or moieties. Common examples of monosaccharides include glucose(dextrose), fructose, galactose, mannose, and ribose. Monosaccharidescan be classified according to the number of carbon atoms of thecarbohydrate, i.e., triose, having 3 carbon atoms such as glyceraldehydeand dihydroxyacetone; tetrose, having 4 carbon atoms such as erythrose,threose and erythrulose; pentose, having 5 carbon atoms such asarabinose, lyxose, ribose, xylose, ribulose and xylulose; hexose, having6 carbon atoms such as allose, altrose, galactose, glucose, gulose,idose, mannose, talose, fructose, psicose, sorbose and tagatose;heptose, having 7 carbon atoms such as mannoheptulose, sedoheptulose;octose, having 8 carbon atoms such as 2-keto-3-deoxy-manno-octonate;nonose, having 9 carbon atoms such as sialose; and decose, having 10carbon atoms.

The above monosaccharides encompass both D- and L-monosaccharides.

In one embodiment, the monosaccharide is a hexose or a hexosederivative. In some embodiments, the hexose is a D-hexose.

In an alternative embodiment, the hexose is a L-hexose.

Alternatively, the monosaccharide can be a monosaccharide derivative, inwhich the saccharide unit comprises one or more substituents other thanhydroxyls. Such derivatives can be, but are not limited to, ethers,esters, amides, acids, phosphates and amines. Amine derivatives include,for example, glucosamine, galactosamine, fructosamine and mannosamine.Amide derivatives include, for example, N-acetylated amine derivativesof saccharides (e.g., N-acetylglucosamine, N-acetylgalactosamine).

Exemplary monosaccharide derivatives include mannose-6-phosphate (M6P),a phosphate derivative, and N-acetylneuraminic acid (a sialic acid), anacid and amide derivative.

Monosaccharides of particular interest include mannose and M6P,interacting specifically with mannose receptors and CI-MPR(cation-independent mannose phosphate receptor) and/or CD-MPR(cation-dependent mannose phosphate receptor), respectively. Thesemonosaccharides are found in natural glycosides on the surface of avariety of lysosomal enzymes, enabling their trafficking to lysosomes,where they exert their specific hydrolytic activity. Othermonosaccharides of particular interest are sialic acids, such asN-acetylneuraminic acid.

As used herein, the phrase “sialic acid” describes an N— or O—derivative of neuraminic acid (in which either the N atom or the O atomof the neuraminic acid are derivatized). N-acetylneuraminic acid is anexemplary sialic acid.

As used herein, the term “M6P” describes the naturally-occurringD-mannose-6-phosphate, as well as the non-naturally occurringL-mannose-6-phsophate, although the first is preferred.

In some embodiments, the linker is attached to the saccharide moiety atthe anomeric carbon (e.g., C-1 in mannose and M6P, C-2 in sialic acid)of the saccharide moiety through an ether bond. The anomeric carbon ofmonosaccharides is typically in the form of acyclic hemiketal orhemiacetal. Thus, an ether bond to an anomeric carbon describes an alkylgroup (e.g., being a part of the linker) bound to an oxygen atom whichis bound to the anomeric carbon, thereby forming an acetal or ketal. Insome embodiments, the linker is attached to the anomeric carbon via an αconfiguration, which is an axial conformation in typical D-hexoses(including D-mannose and M6P).

In many naturally-occurring glycosylated proteins (e.g., M6P-proteinconjugates), the terminal saccharide is linked to the penultimatesaccharide via an α configuration. Conjugation of saccharide moietiescorresponding to such terminal saccharides to a linker of aglycosylation reagent via the natural a configuration enables higheraffinity and recognition between the conjugate formed from theglycosylation reagent and the target receptors.

In an exemplary embodiment, the linker is attached via an αconfiguration to D-mannose, M6P or sialic acid (e.g., N-acetylneuraminicacid). Exemplary glycosylating reagents comprising an M6P saccharidemoiety attached to a linker via an α configuration are depicted in FIGS.1A-B and 2A-D, as well as in Scheme 1 hereinbelow. Exemplaryglycosylating reagents comprising a N-neuraminic acid saccharide moietyattached to a linker via an α configuration are depicted in FIGS. 3A-B.

The linker in the compounds described herein is aimed at linking thesaccharide moiety to a target biomolecule and hence is preferablyselected so as to mimic the structure and chemical characteristics ofnatural glycosylation groups, composed of oligosaccharide chains.

The linker is characterized as having a non-hydrophobic nature due tothe poor functionality exhibited by hydrophobic linkers. Thus, forexample, hydrocarbon chains are not suitable since they are ofhydrophobic nature and are prone to hydrophobic folding, diminishing theexposure of the saccharides to the relevant receptors and evenpreventing interaction between the biomolecule and such receptors.

Optionally, the non-hydrophobic linker is amphiphilic. As used herein,the term “amphiphilic” describes a compound or moiety that issubstantially soluble in both water and a water-immiscible solvent.Amphiphilic linkers can thus avoid folding in both an aqueous andnon-aqueous (e.g., lipid) surroundings, thereby maintaining maximalexposure of the saccharide moiety of the formed conjugate to therelevant receptors.

In some embodiments, the linker is a non-saccharide linker.

A saccharide linker attached to the saccharide moiety would result in adisaccharide, trisaccharide or oligosaccharide moiety. Such groups wouldclosely mimic natural glycosylation groups, as discussed hereinabove,but would be sensitive to enzymes which hydrolyze natural glycosylationgroups. A non-saccharide linker is less prone to hydrolysis, and hence,a glycosylation reagent with a non-saccharide linker provides a longerlasting glycosylated biomolecule in a biological environment.

In some embodiments, the linker comprises a poly(alkylene glycol) chain.

The phrase “poly(alkylene glycol)”, as used herein, encompasses a familyof polyether polymers which share the following general formula:—O—[(CH₂)m-O-]n-, wherein m represents the number of methylene groupspresent in each alkylene glycol unit, and n represents the number ofrepeating units, and therefore represents the size or length of thepolymer. For example, when m=2, the polymer is referred to as apolyethylene glycol, and when m=3, the polymer is referred to as apolypropylene glycol.

In some embodiments, m is an integer greater than 1 (e.g., m=2, 3, 4,etc.).

Optionally, m varies among the units of the poly(alkylene glycol) chain.For example, a poly(alkylene glycol) chain may comprise both ethyleneglycol (m=2) and propylene glycol (m=3) units linked together.

The phrase “poly(alkylene glycol)” also encompasses analogs thereof, inwhich the oxygen atom is replaced by another heteroatom such as, forexample, S, —NH— and the like. This term further encompasses derivativesof the above, in which one or more of the methylene groups composing thepolymer are substituted. Exemplary substituents on the methylene groupsinclude, but are not limited to, alkyl, cycloalkyl, alkoxy, hydroxy,thiol, amine, halo, oxo, carbonyl, carboxylate, carbamate, and the like.

Thus, the phrase “alkylene glycol unit”, as used herein, encompasses a—(CH₂)m-O— group or an analog thereof, as described hereinabove, whichforms the backbone chain of the poly(alkylene glycol), wherein the(CH₂)m (or analog thereof) is bound to a heteroatom belonging to anotheralkylene glycol unit or to a saccharide moiety (in cases of a terminalunit), and the 0 (or heteroatom analog thereof) is bound to the (CH₂)m(or analog thereof) of another alkylene glycol unit, to a reactive group(also referred to herein as “Z”), or to another group at the end ofanother poly(alkylene glycol) chain.

It is to be noted that a heteroatom linking an alkylene glycol unit tothe reactive group Z may optionally be considered as being shared by thealkylene glycol and Z (i.e., belonging to both groups).

An alkylene glycol unit may be branched, such that it is linked to 3 ormore neighboring alkylene glycol units, wherein each of the 3 or moreneighboring alkylene glycol units are part of a poly(alkylene glycol)chain. Such a branched alkylene glycol unit is linked via the heteroatomthereof to one neighboring alkylene glycol unit, and heteroatoms of theremaining neighboring alkylene glycol units are each linked to a carbonatom of the branched alkylene glycol unit. In addition, a heteroatom(e.g., nitrogen) may bind more than one carbon atom of an alkyleneglycol unit of which it is part, thereby forming a branched alkyleneglycol unit (e.g., [(—CH₂)m]₂N— and the like).

In exemplary embodiments, at least 50% of alkylene glycol units areidentical, e.g., they comprise the same heteroatoms and the same mvalues as one another. Optionally, at least 70%, optionally at least90%, and optionally 100% of the alkylene glycol units are identical. Inexemplary embodiments, the heteroatoms bound to the identical alkyleneglycol units are oxygen atoms. In further exemplary embodiments, m is 2for the identical units.

The length of the linker chain is preferably selected so as to conformto the length of the natural glycosyl chains of proteins. Thus,short-chained linkers, such as, for example, poly(ethylene glycol)including up to 4 ethylene glycol units (which form a chain of up to 12atoms in length), are not suitable for enhancing the interaction betweena biomolecule to carbohydrate-specific receptors, such as the CI-MPR.

Thus, in some embodiments, the linker is a poly(alkylene glycol) thatcomprises a chain of at least 18 atoms in length. Accordingly, in anexemplary embodiment, the linker comprises at least 6 ethylene glycolunits. Thus, the poly(alkylene glycol) can comprise 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 and so on, units, as definedhereinabove.

Preferably, the poly(alkylene glycol) comprises a chain of 18 to 170atoms. In some embodiments, the poly(alkylene glycol) comprises a chainof 18 to 100 atoms. In some embodiments, the poly(alkylene glycol)comprises a chain of 18 to 60 atoms. In some embodiments, thepoly(alkylene glycol) comprises a chain of 18 to 36 atoms. In someembodiments, the poly(alkylene glycol) comprises a chain of 24 to 36atoms.

The length of a poly(alkylene glycol) chain, in atoms, is equal ton(m+1), as these variables are defined hereinabove (if m varies amongthe units in the poly(alkylene glycol), the value n(m+1) is based on theaverage value of m).

In one embodiment, the linker is a single, straight chain linker,preferably being polyethylene glycol (PEG).

As used herein, the term “poly(ethylene glycol)” describes apoly(alkylene glycol), as defined hereinabove, wherein at least 50%, atleast 70%, at least 90%, and preferably 100%, of the alkylene glycolunits are —CH₂CH₂—O—. Similarly, the phrase “ethylene glycol units” isdefined herein as units of —CH₂CH₂O—.

The linker may also include branched chains, also known asmulti-antennary. In such branched linkers, each branched chain is linkedto a saccharide moiety, and the variable X in the above formularepresents all saccharide moieties bound to the linker. Bi-, tri- ormulti-antennary linkers may be used to enhance the binding to specificreceptors.

Glysosylation reagents having a branched linker can therefore berepresented by the general formula:

(X)n-A-Z

wherein X and Z are as described herein, A is a branched linker asdefined herein and n corresponds to the number of branched chains of thebranched linker.

It is to be understood that the length, in atoms, of a poly(alkyleneglycol) or a linker, refers to the number of atoms forming a linearchain between the saccharide moiety and the reactive group (e.g., refersto the variable A in the formula hereinabove). Thus, a branched linkerwhich links a plurality of saccharide moieties to a reactive group willbe characterized by a plurality of lengths, one length for eachsaccharide moiety, whereby the length of each linker is as definedherein (e.g., at least 18 atoms in length).

A branched linker allows the attachment of a plurality of saccharidemoieties via a single site on a biomolecule. Exemplary glycosylatingreagents comprising a branched linker are depicted in FIGS. 2A-D. Theuse of a branched linker is especially beneficial in case thebiomolecule exhibits a limited number of potential anchoring positionsor in case the attachment of a small number of linkers per biomoleculeis desired. A branched linker glycosylation reagent may also be used tomimic the structure of natural branched oligosaccharide moieties (e.g.,in glycosylated proteins) which comprise two or more terminalsaccharides (e.g., M6P) per oligosaccharide moiety. Such branchedoligosaccharide moieties may be characterized by a higher affinity to areceptor of the terminal saccharide.

In an optional embodiment, the linker comprises at least two chemicalmoieties which are covalently linked to one another. For example, thelinker may comprise two or more poly(alkylene glycol) moieties whichhave been covalently linked to one another so as to form a longer linearpoly(alkylene glycol) moiety from shorter poly(alkylene glycol) moietiesand/or so as to form a branched poly(alkylene glycol) moiety from linearpoly(alkylene glycol) moieties.

The chemical moieties linked to one another may be the same or differentfrom one another.

The chemical nature of the linkage between such moieties depends on thereactive group(s) present within the poly(alkylene glycol) moieties, andcan be, for example, an ester, an amide, an amine, an ether, athioether, a disulfide, a sulfonate, a sulfonyl, a sulfinyl, aphosphate, a phosphonate, a phosphinyl, a carbamate, a thiocarbamate, aurea, a thiourea, a thiocarboxylate, a hydrazine, azo, a guanidinogroup, a guanyl and the like, all being linking groups linking the twopoly(alkylene glycol) moieties.

An elongated linker may be prepared from two linker moieties (e.g.,poly(alkylene glycol) moieties, as defined herein), wherein one linkermoiety comprises a first reactive group (as defined herein) and a secondreactive group, and the other linker moiety comprises a third reactivegroup, wherein the second and third reactive groups on the moieties canbe reacted with each other so as to form a linkage, as describedhereinabove. For example, the second and third reactive groups may be acarboxylate and an amine, such that they react to form an amide linkage.The elongated linker thus formed comprises the first reactive group (theZ moiety referred to herein).

Similarly, an elongated linker may be prepared from more than two linkermoieties, using the two moieties described hereinabove in combinationwith one or more moieties having two reactive groups.

Optionally, the elongated linker is prepared with an attached saccharidemoiety. In such a case, the aforementioned linker moiety comprising thethird reactive group is attached to a saccharide moiety.

Alternatively, the elongated linker is prepared without an attachedsaccharide moiety. The saccharide moiety is then attached to theelongated linker in order to obtain the glycosylating reagent. In suchcases, the linker moiety which is aimed at being attached to thesaccharide moiety (e.g., the aforementioned linker moiety comprising thethird reactive group) can be protected in order to avoid a reaction ofthis portion of the polymer.

A branched linker may be prepared as described hereinabove for anelongated linker, except that at least one linker moiety has additionalreactive groups capable of forming linkages.

For example, a moiety comprising a first reactive group and/or a moietyattached to a saccharide (or capable of being attached to a saccharidemoiety) may further comprise two or more additional reactive groups,rather than one. Thus, for example, a linker moiety comprising a firstreactive group and two or more carboxylate groups may be attached to anamine group of each of two or more linker moieties attached to asaccharide moiety, thereby forming an elongated linker with a firstreactive group and a plurality of amide linkage, which is attached to aplurality of saccharide moieties.

Alternatively, one or more branching moieties may comprise three or morereactive groups. For example, a linker moiety comprising a firstreactive group and a second reactive group is attached to a branchingmoiety by reacting the second branching moiety with a reactive group onthe branching moiety so as to form a linkage. The remaining reactivegroups of the branching moiety are each reacted with a third reactivegroup on a linker moiety attached to a saccharide moiety (or capable ofbeing attached to a saccharide moiety).

Optionally, the branching moiety comprise a single reactive group (e.g.,amine) capable of linking to the second reactive group (e.g.,carboxylate) of a moiety comprising a first and second reactive group,and a plurality of reactive groups (e.g., carboxylates) capable oflinking to a third reactive group (e.g., amine) of a linker moietyattached to a saccharide (or capable of being attached to a saccharidemoiety). This facilitates the preparation of a branched glycosylatingreagent comprising a single reactive group (i.e., the first reactivegroup described herein) and a plurality of saccharide moieties.

The linker comprises a reactive group that forms a part thereof, alsoreferred to herein as “Z”. The linker preferably terminates by thereactive group. A linker is considered terminated by the reactive groupwhen the reactive group is located at the portion of the linker that isfarthest from the saccharide moiety.

The reactive group is preferably selected to as to enable itsconjugation to biomolecules. Exemplary reactive groups include, but arenot limited to, carboxylate (e.g., —CO₂H), thiol (—SH), amine (—NH₂),halo, azide (—N₃), isocyanate (—NCO), isothiocyanate (—N═C═S), hydroxy(—OH), carbonyl (e.g., aldehyde), maleimide, sulfate, phosphate,sulfonyl (e.g. mesyl, tosyl), etc. as well as activated groups, such asN-hydroxysuccinimide (NHS) (e.g. NHS esters),sulfo-N-hydroxysuccinimide, anhydride, acyl halide (—C(═O)-halogen) etc.

The reactive groups of the linker may be in a protected form thereof.

As used herein, the phrase “protected form” describes a derivative of achemical group (e.g., a reactive group) which is less reactive than thechemical group, wherein the derivative can be readily reacted so as torevert to the original chemical group.

A reactive group may be protected by attaching thereto a protectinggroup. Many adequate protecting groups will be known to one of skill inthe art. The latter should be removed prior to conjugation to the targetbiomolecule. A suitable protecting group is selected such that it can bereadily removed without affecting other linkages or functionalities inthe molecule (e.g., the conjugate or glycosylation reagent as describedherein).

For example, hydroxy groups may be protected in the form of acarboxylate by attaching thereto a carbonyl group (e.g., acetyl), in theform of an ether (e.g., methoxymethyl ether, p-methoxybenzyl ether,methylthiomethyl ether) or by attaching a silyl group (e.g.,trialkylsilyl).

Amine groups may be protected, for example, in the form of an amide(e.g., by attaching a carbobenzyloxy group, t-butoxycarbonyl group, or9-fluorenylmethyloxycarbonyl group).

Carboxylic acid groups, for example, may be protected in the form ofesters (e.g., methyl, benzyl, t-butyl esters).

Carbonyl groups, for example, may be protected in the form of acetals(e.g., for protecting aldehydes) or ketals (e.g., for protectingketones).

Alternatively, the reactive group may be in an activated form thereof.

As used herein, the phrase “activated form” describes a derivative of achemical group (e.g., a reactive group) which is more reactive than thechemical group, and which is thus readily capable of undergoing achemical reaction that leads to a bond formation. The activated form maycomprise a particularly suitable leaving group, thereby facilitatingsubstitution reactions. For example, a —C(═O)—NHS group(N-hydroxysuccinimide ester, or —C(═O)—O-succinimide) is a well-knownactivated form of —C(═O)OH, as NHS (N-hydroxysuccinimide) can be reactedwith a —C(═O)OH to form —C(═O)—NHS, which readily reacts to formproducts characteristic of reactions involving —C(═O)OH groups, such asamides and esters.

The reactive group can be attached to the linker via different groups,atoms or bonds. These may include an ether bond [e.g., —O-alkyl-], anester bond [e.g., —O—C(═O)-alkyl-], a carbamate [e.g.,O—C(═O)—NH-alkyl-], etc. Thus, a variety of terminal groups can beemployed.

The following are non-limiting examples of the different groups that mayconstitute the non-saccharide end of the linker chain: Z=—CH₂CO₂H,—CH₂CH₂CO₂H, —CH₂CH₂SH, —CH₂CH₂NH₂, —CH₂CH₂N₃, —CH₂CH₂NCO, —CH₂—COO—NHS,—CH₂CH₂—COO—NHS, —CO—CH₂—COO—NHS, —CH₂CH₂—NHCOCH₂CH₂-maleimide, etc.

The number of methylene groups in each of the above reactive groups ismerely exemplary, and may be varied.

The reactive group may also comprise the heteroatom at the end of apoly(alkylene glycol) chain (e.g., —OH).

In exemplary embodiments of the present invention, the reactive group isselected from the group consisting of an amine, a maleimide and acarboxylate, including protected forms and activated forms thereof, asthese terms are defined herein. In exemplary embodiments, thecarboxylate is a C-carboxy group such as a carboxylic acid, as theseterms are defined herein.

As used herein, the terms “amine” and “amino” refer to either a —NR′R″group, wherein R′ and R″ are selected from the group consisting ofhydrogen, alkyl, cycloalkyl, heteroalicyclic (bonded through a ringcarbon), aryl and heteroaryl (bonded through a ring carbon). R′ and R″are bound via a carbon atom thereof. Optionally, R′ and R″ are selectedfrom the group consisting of hydrogen and alkyl comprising 1 to 4 carbonatoms. Optionally, R′ and R″ are hydrogen.

As used herein throughout, the term “alkyl” refers to a saturatedaliphatic hydrocarbon including straight chain and branched chaingroups. Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever anumerical range; e.g., “1-20”, is stated herein, it implies that thegroup, in this case the alkyl group, may contain 1 carbon atom, 2 carbonatoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. Morepreferably, the alkyl is a medium size alkyl having 1 to 10 carbonatoms. Most preferably, unless otherwise indicated, the alkyl is a loweralkyl having 1 to 4 carbon atoms. The alkyl group may be substituted orunsubstituted. When substituted, the substituent group can be, forexample, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide,phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as these termsare defined herein.

A “cycloalkyl” group refers to an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereinone of more of the rings does not have a completely conjugatedpi-electron system. Examples, without limitation, of cycloalkyl groupsare cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane. Acycloalkyl group may be substituted or unsubstituted. When substituted,the substituent group can be, for example, alkyl, alkenyl, alkynyl,aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro,azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea,thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as theseterms are defined herein.

An “alkenyl” group refers to an alkyl group which consists of at leasttwo carbon atoms and at least one carbon-carbon double bond.

An “alkynyl” group refers to an alkyl group which consists of at leasttwo carbon atoms and at least one carbon-carbon triple bond.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. Examples,without limitation, of aryl groups are phenyl, naphthalenyl andanthracenyl. The aryl group may be substituted or unsubstituted. Whensubstituted, the substituent group can be, for example, alkyl, alkenyl,alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy,alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl,sulfonyl, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl,thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, andamino, as these terms are defined herein.

A “heteroaryl” group refers to a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl groups include pyrrole, furane,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline and purine. The heteroaryl group may besubstituted or unsubstituted. When substituted, the substituent groupcan be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro,azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea,thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as theseterms are defined herein.

A “heteroalicyclic” group refers to a monocyclic or fused ring grouphaving in the ring(s) one or more atoms such as nitrogen, oxygen andsulfur. The rings may also have one or more double bonds. However, therings do not have a completely conjugated pi-electron system. Theheteroalicyclic may be substituted or unsubstituted. When substituted,the substituted group can be, for example, lone pair electrons, alkyl,alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo,hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy,sulfinyl, sulfonyl, cyano, nitro, azide, phosphonyl, phosphinyl, oxo,carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,sulfonamido, and amino, as these terms are defined herein.Representative examples are piperidine, piperazine, tetrahydro furane,tetrahydropyrane, morpholino and the like.

A “hydroxy” group refers to an —OH group.

An “azide” group refers to a —N═N⁺═N⁻ group.

An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group,as defined herein.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group,as defined herein.

An “ether” refers to both an alkoxy and an aryloxy group, wherein thegroup is linked to an alkyl, alkenyl, alkymyl, cycloalkyl, aryl,heteroaryl or heteroalicyclic group.

A “thiohydroxy” or “thiol” group refers to a —SH group.

A “thioalkoxy” group refers to both an —S-alkyl group, and an—S-cycloalkyl group, as defined herein.

A “thioaryloxy” group refers to both an —S-aryl and an —S-heteroarylgroup, as defined herein.

A “thioether” refers to both a thioalkoxy and a thioaryloxy group,wherein the group is linked to an alkyl, alkenyl, alkymyl, cycloalkyl,aryl, heteroaryl or heteroalicyclic group.

A “disulfide” group refers to both a —S-thioalkoxy and a —S-thioaryloxygroup.

A “carbonyl” group refers to a —C(═O)—R′ group, where R′ is defined ashereinabove.

An “aldehyde” group refers to a carbonyl group, where R′ is hydrogen.

A “thiocarbonyl” group refers to a —C(═S)—R′ group, where R′ is asdefined herein.

A “C-carboxy” group refers to a —C(═O)—O—R′ groups, where R′ is asdefined herein.

An “O-carboxy” group refers to an R′C(═O)—O— group, where R′ is asdefined herein.

An “oxo” group refers to a ═O group.

A “carboxylate” encompasses both C-carboxy and O-carboxy groups, asdefined herein.

A “carboxylic acid” group refers to a C-carboxy group in which R′ ishydrogen.

A “thiocarboxy” or “thiocarboxylate” group refers to both —C(═S)—O—R′and —O—C(═S)R′ groups.

An “ester” refers to a C-carboxy group wherein R′ is not hydrogen.

A “halo” group refers to fluorine, chlorine, bromine or iodine.

A “sulfinyl” group refers to an —S(═O)—R′ group, where R′ is as definedherein.

A “sulfonyl” group refers to an —S(═O)₂—R′ group, where R′ is as definedherein.

A “sulfonate” group refers to an —S(═O)₂—O—R′ group, where R′ is asdefined herein.

A “sulfate” group refers to an —O—S(═O)₂—O—R′ group, where R′ is asdefined as herein.

A “sulfonamide” or “sulfonamido” group encompasses both S-sulfonamidoand N-sulfonamido groups, as defined herein.

An “S-sulfonamido” group refers to a —S(═O)₂—NR′R″ group, with each ofR′ and R″ as defined herein.

An “N-sulfonamido” group refers to an R′S(═O)₂—NR″ group, where each ofR′ and R″ is as defined herein.

An “O-carbamyl” group refers to an —OC(═O)—NR′R″ group, where each of R′and R″ is as defined herein.

An “N-carbamyl” group refers to an R′OC(═O)—NR″— group, where each of R′and R″ is as defined herein.

A “carbamyl” or “carbamate” group encompasses O-carbamyl and N-carbamylgroups.

An “O-thiocarbamyl” group refers to an —OC(═S)—NR′R″ group, where eachof R′ and R″ is as defined herein.

An “N-thiocarbamyl” group refers to an R′OC(═S)NR″— group, where each ofR′ and R″ is as defined herein.

A “thiocarbamyl” or “thiocarbamate” group encompasses O-thiocarbamyl andN-thiocarbamyl groups.

A “C-amido” group refers to a —C(═O)—NR′R″ group, where each of R′ andR″ is as defined herein.

An “N-amido” group refers to an R′C(═O)—NR″— group, where each of R′ andR″ is as defined herein.

An “amide” group encompasses both C-amido and N-amido groups.

A “urea” group refers to an —N(R′)—C(═O)—NR″R′″ group, where each of R′and R″ is as defined herein, and R′″ is defined as R′ and R″ are definedherein.

A “guanidino” group refers to an —N(R′)C(═N—R″)—NR′″R″″ group, whereeach of R′, R″ and R′″ is as defined herein, and R″″ is defined as R′and R″ are defined herein.

A “guanyl” group refers to an R′R″NC(═N—R′″)— group, where each of R′,R″ and R′″ is as defined herein.

A “nitro” group refers to an —NO₂ group.

A “cyano” group refers to a —C≡N group.

The term “phosphonyl” or “phosphonate” describes a —P(═O)(OR′)(OR″)group, with R′ and R″ as defined hereinabove.

The term “phosphate” describes an —O—P(═O)(OR′)(OR″) group, with each ofR′ and R″ as defined hereinabove.

A “phosphoric acid” is a phosphate group is which each of R is hydrogen.

The term “phosphinyl” describes a —PR′R″ group, with each of R′ and R″as defined hereinabove.

The term “thiourea” describes a —N(R′)—C(═S)—NR″— group, with each of R′and R″ as defined hereinabove.

A “hydrazine” group refers to a —N(R′)—NR″R′″ group.

An “azo” group refers to a —N═NR′ group.

As used herein, the term “maleimide” refers to a cyclic—[NC(═O)—C(R′)=C(R″)—C(═O)]— moiety, which is linked via the N atom, andwherein R′ and R″ are as defined hereinabove. The above definitionsdescribe groups as monovalent radicals, i.e., they attach to a singlemoiety. However, as used herein, many of the groups are intended toencompass groups that attach to a plurality of moieties (“linkinggroups”), thereby linking the moieties together. Specifically, alkyl,cycloalkyl, aryl and heteroaryl, as defined herein, may attach to morethan one moiety. In addition, each group defined above as comprising anyof R′, R″, R′″ and R″″, may alternatively be defined as a linking groupwherein at least one R′, R″, R′″ or R″″, as these terms are definedherein, is a moiety attached to the linking group, rather than acomponent of the linking group (e.g., the amine group —NR′R″ may bedefined as a group linking a first moiety, an R′ moiety, and an R″moiety).

In an optional embodiment the glycosylation reagent has the formuladepicted in Scheme 1.

wherein:

n>6; and

Z is selected from the group consisting of —CH₂CO₂H, —CH₂CH₂CO₂H,—CH₂CH₂SH, —CH₂CH₂NH₂, —CH₂CH₂N₃, —CH₂CH₂NCO, —CH₂—COO—NHS,—CH₂CH₂—COO—NHS, —CO—CH₂—COO—NHS, and —CH₂CH₂—NHCOCH₂CH₂-maleimide(wherein —CH₂CH₂—NHCOCH₂CH₂— is bound to the nitrogen atom ofmaleimide).

Although the above formula depicts the phosphate group as a sodium salt,all formulas herein which describe a phosphate group in any form are tobe understood as encompassing the acid form (—P(═O)(OH)₂) as well as allsalts thereof, including salts comprising one or two cations, and saltsincluding cations other than sodium, unless stated otherwise.

According to an exemplary embodiment of the present invention, n=8 and Zis —CH₂CH₂CO₂H. This compound is also referred to herein asM6P-PEG₈-CO₂H or M6P-PEG₈-COOH.

According to another exemplary embodiment of the present invention, n=8and Z is —CH₂CH₂—NHCOCH₂CH₂-maleimide (see FIG. 1B). This compound isalso referred to herein as M6P-PEG₈-maleimide.

Another exemplary glycosylation reagent according to scheme 1, whereinn=12 and Z is —CH₂CH₂CO₂H, is depicted in FIG. 1A. This compound is alsoreferred to herein as M6P-PEG₁₂-CO₂H or M6P-PEG₁₂-COOH.

According to further exemplary embodiments of the present invention, theglycosylating reagent has the formula depicted in FIG. 2A, 2B, 2C, 2D,3A or 3B.

The reagents of the invention can be synthesized in various ways,employing general synthetic methodologies. Exemplary methods arepresented hereinbelow but other techniques, methodologies or reagentscan be employed or envisioned by those skilled in the art.

In general, a glycosylation reagent according to the present embodimentscan be prepared by treating a saccharide with a HO-A-Z derivative,wherein A and Z are defined as hereinabove. Groups other than the —OHgroup (e.g., the Z reactive group) can be pre-protected in order toavoid their reaction with the mono-saccharide. In cases where thechemical reactivity of the non-hydroxyl groups (Z) is not expected tolead to an undesired reaction with the saccharide under the reactionconditions of the hydroxyl conjugation to the monosaccharide, priorprotection may be unnecessary. In cases where a reactive group isprotected, it should be de-protected prior to its use for conjugation tothe target biomolecule. If a group other than a reactive group isprotected, the group may be de-protected before or after conjugation tothe target biomolecule.

The linker can be attached to the saccharide moiety via varioussynthetic methodologies.

According to one exemplary methodology, penta-acetylated saccharide ismixed with a mono-protected linker (e.g., wherein Z comprises acarboxylic acid protected as an ester) in the presence of borontrifluoride etherate to give an adduct of the linker and the acetylatedmonosaccharide. Deprotection with aqueous sodium hydroxide gives aglycosylating reagent X-A-Z, wherein Z comprises a carboxylic acidgroup. This carboxylic acid group can be further used for conjugation toproteins as described below.

Alternatively, as exemplified in Example 1, the penta-acetylatedsaccharide is reacted with mono-protected linker in the presence ofZnCl₂, under elevated temperature conditions (100-110° C.) and vacuum.The product solution is washed with water, the organic solvent isevaporated under reduced pressure and the crude product is purified bycrystallization or chromatography of Silica-Gel. The acetyl protectinggroups are easily removed by sodium methoxide in methanol.

The use of a penta-acetylated saccharide, as described hereinabove,refers to a saccharide, typically a hexose, having 4 free hydroxylgroups other than the hydroxyl group which is intended to react with thelinker (e.g., the free hydroxyl attached to the anomeric carbon). Thenumber of protecting groups (e.g., acetyls) which should be attached tothe saccharide according to the procedures described herein will dependon the saccharide being used (e.g., M6P comprises 3 free hydroxyl groupsother than the hydroxyl group which is intended to react with thelinker).

It is to be noted that in the aforementioned example, acetylation of thehydroxyl attached to the anomeric carbon does not prevent its reactionwith the linker.

The carboxylate reactive group of linkers such as in the proceduredescribed above can also be activated by various methods, to enable easycoupling to proteins or other biomolecules. This carboxylic group can beconverted to activated carboxamides by the use ofN,N,N′,N″-tetramethyl(succinimido)uronium tetrafluoroborate (TSTU). Thisactivated carboxamide can be easily used for conjugation to proteins atmild alkaline pH.

A carboxylate reactive group may also be activated by preparing an NHSester, for example, by activating the corresponding carboxylic acid byusing NHS and a carbodiimide agent, such as DCC, optionally catalyzed bya proton scavenger such as dimethylaminopyridine (DMAP). Alternatively,EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) isused as the carbodiimide agent. Excess reagents (e.g., DCC, EDC, NHS)and/or byproducts may optionally be separated using an appropriatechromatographic column or through selective crystallization.

As shown in the Examples section that follows, the amount of theactivating agents, which form that activated form of the reactive group,utilized can affect the properties of the resulting conjugate (formedupon conjugating the glycosylation reagent to the biomolecule).

The activation of the carboxylate reactive group can optionally beperformed prior to the conjugation of the linker to a saccharide moiety.A glycosylating reagent can also be prepared by treating a targetsaccharide with a HO-A-Z compound under mildly acidic conditions. Thehydroxyl group reacts with the anomeric carbon to give a glycosylatingreagent. This methodology usually results in a mixture of anomers. Theseanomers can be further separated by selective crystallization,chromatography or additional common separation techniques.

The glycosylating reagent can also be synthesized by utilizing enzymaticcatalysts, preferably immobilized on non-soluble supports, underaqueous, non-aqueous or micro-aqueous conditions. This methodology mayemploy different commercially available glycosidases as the catalyst.

As discussed hereinabove, the glycosylating reagents described hereinare designed suitable for being conjugated to a target biomolecule andare particularly aimed at introducing a saccharide moiety to thebiomolecule, for example, so as to beneficially affect its traffickingand uptake by targeted cells, tissues and/or organelles.

Herein, a conjugate formed by reacting a biomolecule with aglycosylating reagent having the formula X-A-Z, as defined hereinabove,is described as having a formula X-A-W—B, wherein B is the conjugatedbiomolecule, W is the chemical group obtained by the reaction of thereactive group Z with the biomolecule, and X and A are defined as forthe glycosylating reagent.

Thus, for example, the glycosylating reagent depicted in scheme 1hereinabove reacts with a biomolecule to form a conjugate having theformula depicted in Scheme 2.

wherein:

n>6;

W is a chemical group obtained by the reaction of the reactive group Z,as defined in Scheme 1, with the biomolecule; and

B is the conjugated biomolecule.

The glycosylation reagents of the invention may be used to glycosylate avariety of biomolecules, such as proteins, peptides, oligonucleotides,antisense molecules, polynucleotides, steroids, antibodies, antigens,toxins, growth factors, agonists, antagonists, co-factors, cytokines,enzymes, immunoglobulins, hormones, inhibitors, ligands, prostaglandins,vaccines and vitamins.

The biomolecules can be, for example, derived from eukaryotic (includinganimal, plant, yeast and fungal), mammalian, human, prokaryotic,bacterial or viral source.

The terms “polypeptide” and “protein”, which are used hereininterchangeably, refer to a polymeric form of amino acids of 10 or more,and more preferably 100 or more amino acids, which can include coded andnon-coded amino acids, chemically or biochemically modified orderivatized amino acids, and polypeptides having modified peptidebackbones. Polypeptides may be polymers of naturally occurring aminoacid residues; non-naturally occurring amino acid residues, such as, forexample N-substituted glycine residues, amino acid substitutes, and thelike; and both naturally occurring and non-naturally occurring aminoacid residues/substitutes. This term does not refer to or excludespost-translational modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. The termincludes ribosomally or synthetically made polypeptides, fusionproteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusions with heterologous andhomologous leader sequences; immunologically tagged proteins; and thelike.

The term “peptide” is defined as is the term “polypeptide” herein, butrefers to compounds comprising 2-9 amino acids, rather than compoundshaving 10 or more amino acids.

As used herein throughout the term “amino acid” or “amino acids” isunderstood to include the 20 genetically coded or naturally occurringamino acids; those amino acids often modified post-translationally invivo, including, for example, hydroxyproline, phosphoserine andphosphothreonine; and other unusual amino acids including, but notlimited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,nor-leucine and ornithine. Furthermore, the term “amino acid” includesboth D- and L-amino acids and other non-naturally occurring amino acids.

In some embodiments of the present invention, the biomolecule is aprotein, including, for example, hormones, growth factors, enzymes,antibodies, chimeric proteins, structural protein, binding proteins,blood factors and the like. Enzymes are exemplary proteins suitable forinclusion in embodiments of the present invention.

The protein may be a naturally produced protein isolated from abiological source, a synthetically-prepared protein, synthesized (e.g.,from amino acids) according methods known in the chemical arts, or arecombinant protein (e.g., produced via genetic engineering). Arecombinant protein may be produced, for example, in host cells such asbacterial cells, in fungal cells, in yeast, in whole plants, in plantcells, in mammalian products (e.g., urine, milk) and/or in mammaliancells.

In some embodiments, the protein is recombinantly produced innon-mammalian cells such as, for example, plant cells.

In an exemplary embodiment, the glycosylation reagents of the inventionare used to modify lysosomal proteins (e.g. enzymes), such that thebiomolecule is a lysosomal protein.

Exemplary lysosomal proteins which may be modified according toembodiments of the present invention include, without limitation,lysosomal protective protein, L-iduronidase, iduronate-2-sulfatase,heparan-N-sulfatase, α-N-acetylglucosaminidase,acetylCoA:α-glucosaminide acetyltransferase,N-acetylglucosamine-6-sulfatase, galactose-6-sulfatase, β-galactosidase,N-acetylgalactosamine-4-sulfatase, β-glucuronidase,hyaluronoglucosaminidase, aspartylglucosaminidase, acid lipase, cystinetransporter, Lamp-2, α-galactosidase A, acid ceramidase, α-L-fucosidase,glucocerebrosidase, galactocerebrosidase, α-glucosidase,β-galactosidase, β-hexosaminidase A, β-hexosaminidase B, ganglioside GM2activator protein, α-D-mannosidase, β-D-mannosidase, arylsulfatase A,saposin B, neuraminidase, phosphotransferase, palmitoyl proteinthioesterase, tripeptidyl peptidase I, acid sphingomyelinase, cathepsinK, α-galactosidase B, sialic acid transporter, tartrate-resistant acidphosphatase, asparaginase, ceroid lipofuscinosis neuronal protein 5,CPVL, cathepsin B, dipeptidyl-peptidase I, cathepsin D, cathepsin H,cathepsin L, cathepsin S, cathepsin Z, deoxyribonuclease II,dipeptidyl-peptidase II, N-acetylgalactosamine-6-sulfatase, γ-glutamylhydrolase, heparanase, legumain, 1-O-acylceramide synthase,myeloperoxidase, α-N-acetylgalactosaminidase, NPC2 protein, plasmaglutamate carboxypeptidase, Pro-X carboxypeptidase, proactivatorpolypeptide, N-sulfoglucosamine sulfohydrolase, sialic acid9-O-acetylesterase, and tripeptidyl-peptidase I.

Optionally, the lysosomal enzyme is modified by being linked to a M6Pmoiety. Without being bound by any particular theory, it is believedthat the attachment of a M6P moiety facilitates the trafficking ofproteins into lysosomes. The trafficking of lysosomal proteins intolysosomes is typically essential for the proper function of lysosomalproteins. Lysosomal enzymes are typically relatively inactive at the pHfound outside of the lysosome, and in any case, the activity of alysosomal enzyme outside a lysosome will in many cases be harmful.Moreover, the proper function of lysosomal proteins is commonlyessential for the health of an organism. Thus, for example, deficienciesof many lysosomal proteins result in severe and even lethal diseases ordisorders, as discussed in detail hereinbelow.

The conjugation of M6P to a protein is particularly useful for proteinsoriginating from organisms (e.g., microorganisms) or cells (e.g. plantsand plant cells) in which there is no M6P glycosylation of proteins.

Embodiments of the present invention may optionally be used to treat adisease or disorder associated with a deficiency of a lysosomal proteinin lysosomes. Examples of such diseases and disorders include (deficientprotein in parentheses) mucopolysaccharidosis type I (L-iduronase),mucopolysaccharidosis type II (iduronate-2-sulfatase),mucopolysaccharidosis type IIIA (heparan-N-sulfatase),mucopolysaccharidosis type IIIB (a-N-acetylglucosaminidase),mucopolysaccharidosis type IIIC (acetylCoA:α-glucosaminideacetyltransferase), mucopolysaccharidosis type IIID(N-acetylglucosamine-6-sulfatase), mucopolysaccharidosis type IVA(galactose-6-sulfatase), mucopolysaccharidosis type IVB(β-galactosidase), mucopolysaccharidosis type VI(N-acetylgalactosamine-4-sulfatase), mucopolysaccharidosis type VII(β-glucuronidase), mucopolysaccharidosis type IX(hyaluronoglucosaminidase), aspartylglucosaminuria(aspartylglucosaminidase), cholesterol ester storage disease (acidlipase), cystinosis (cystine transporter), Danon disease (Lamp-2), Fabrydisease (α-galactosidase A), Farber disease (acid ceramidase),fucosidosis (α-L-fucosidase), galactosialidosis (lysosomal protectiveprotein), Gaucher disease (glucocerebrosidase), globoid cellleucodystrophy (galactocerebrosidase), GM1-gangliosidosis(β-galactosidase), Tay Sachs disease (β-hexosaminidase A), Sandhoffdisease (β-hexosaminidase A and B), GM2-gangliosidosis (ganglioside GM2activator protein), α-mannosidosis (α-D-mannosidase), β-mannosidosis(β-D-mannosidase), metachromatic leucodystrophy (arylsulfatase A,saposin), mucolipidosis type I (neuraminidase), mucolipidosis type II(phosphotransferase), mucolipidosis type IIIC (phosphotransferase),multiple sulfatase deficiency (sulfatases), CLN1 Batten disease(palmitoyl protein thioesterase), CLN2 Batten disease (tripeptidylpeptidase I), Niemann-Pick disease types A and B (acidsphingomyelinase), Niemann-Pick disease type C, pycnodysostosis(cathepsin K), Schindler disease (α-galactosidase B) and sialic acidstorage disease (sialic acid transporter).

α-Galactosidase (e.g., α-galactosidase A) and glucocerebrosidase areexemplary lysosomal proteins for use as a biomolecule in conjugatesaccording to embodiments of the present invention. As described herein,α-galactosidase A-containing conjugates may be used for treating Fabrydisease, α-galactosidase B-containing conjugates may be used fortreating Schindler disease, and glucocerebrosidase-containing conjugatesmay be used for treating Gaucher's disease.

Alternatively, embodiments of the present invention relate to linkingM6P to other (e.g., non-lysosomal) proteins which have been found to benaturally linked to M6P. Exemplary such proteins include, withoutlimitation, lactotransferrin, pancreatic ribonuclease, hornerin,cation-dependent mannose-6-phosphate receptor, ribonuclease K6,intercellular adhesion molecule 1, CREG1 protein, laminin A, hemoglobinζ chain, cerebellin 4, desmoplakin, fatty acid-binding protein,sulfatase-modifying factor, leukocyte elastase,procollagen-lysine-2-oxoglutarate-5-dioxygenase 1, ferritin light chain,acid sphingomyelinase-like phosphodiesterase 3A, hemoglobin β chain,ribonuclease T2, cat eye syndrome critical region 1, leucine-richα₂-glycoprotein, antithrombin-III, serum amyloid P-component, plasmaserine protease inhibitor, haptoglobin-related protein, complement C1qsubcomponent A chain, complement C1q subcomponent B chain, complementC1q subcomponent C chain, cholinesterase, angiotensinogen,prostaglandin-H₂ D-isomerase, plasma protease Cl inhibitor, mammalianependymin-related protein, α₁B-glycoprotein, plasma kallikrein,hemopexin, AMBP protein, α₁-antitrypsin, pigment epithelium-derivedfactor, α₂-macroglobulin, kallistatin, Fc fragment of IgG-bindingprotein, corticosteroid-binding globulin, zinc-α₂-glycoprotein, afamin,serotransferrin, ceruplasmin, biotimidase, ficolin-3, serum albumin,α₁-acid glycoprotein 1, α₁-acid glycoprotein 2, CD5 antigen-like,complement C2 precursor, complement C3 precursor, inter-α-trypsininhibitor heavy chain H4, inter-α-trypsin inhibitor heavy chain 2,inter-α-trypsin inhibitor heavy chain 1, ficolin-2, complement factor B,dopamine β-hydroxylase, fibrinogen 0 chain, α₁-antichymotrypsin,extracellular matrix protein 1, kininogen-1, lumican, complementcomponent 4B, cation-independent mannose-6-phosphate receptor,adipocyte-derived leucine aminopeptidase, fetuin-B,N-acetylmuramoyl-L-alanine amidase, histidine-rich glycoprotein,vitronectin, α₂-HS-glycoprotein, clusterin, C4b-binding protein α chain,mannan-binding lectin serine protease 1, and transthyretin (Sleat etal., Molecular & Cellular Proteomics Methodologies 5:1942-1956, 2006).

Further alternatively, embodiments of the present invention relate tolinking M6P or any other saccharide to any protein which exhibit atherapeutic activity.

These include, in addition to the above-listed proteins, Factor VII,Factor VIII, Factor IX, Protein C (Serine protease), IFN-beta, IFN-alphaDNase, hyaluronidase, fibrolase, plasminogen activator, BMP (bonemorphogenetic protein), PDGF (platelete derived growth factor) EPO(Erythropoietin), LH (luteinizing hormone), RHCG (Rh family, Cglycoprotein) TNF receptor, IL-1, IL-2, IL-11, urate oxidase, TSH(thyroid stimulating hormone), Glucagon, tPA-(Tissue plasminogenactivator), Insulin, Growth Hormone, calcitonin, GM-CSF(granulocytemacrophage colony-stimulating factor), IGF-1 keratinocyte growth factor,TNF-α, Hirudin, Apo2L, Antithrombin III, kallikrein inhibitor, AAT(alpha-1 antitrypsin), lipase, protease, amylase, and VEGF.

As discussed hereinabove, some embodiments of the present inventioncomprise sialic acid (e.g. N-acetylneuraminic acid) as a saccharidemoiety. Follicle-stimulating hormone (FSH) is an exemplary protein forinclusion in conjugates comprising a sialic acid saccharide moiety.Without being bound by any particular theory, it is believed that sialicacid naturally increases the half-life of a glycosylated protein'scirculation in serum by masking saccharides from receptors. Thus, it isbelieved that conjugation of one or more sialic acid moieties to FSHincreases the half-life of the FSH in serum, thereby enhancing theefficacy of the FSH. The half-life of biomolecules in serum is highlyimportant from a pharmacological aspect, and increasing the half-life istypically imperative in the development of pharmaceutical biomolecule.

In some embodiments of the present invention, a biomolecule which issuitable for use as a labeling agent is conjugated with theglycosylating agent described herein.

As used herein, the term “labeling agent” refers to a molecule which isreadily detected. Exemplary labeling agents include chromophores,fluorophores, chemiluminescent agents, and radiolabeling agents (i.e.,radioactive labeling agents).

Such a molecule is also referred to herein as being “labeled”.Alternatively, a labeling agent may be a molecule that is capable ofbinding by any of the aforementioned labeling agents. Examples of suchmolecules include antigens capable of binding to labeled antibodies,antisense molecules capable of binding to labeled oligonucleotidesand/or polynucleotides, agonists and antagonists capable of binding tolabeled enzymes, ligands capable of binding to labeled receptors and thelike. In addition to the aforementioned examples, the antigen,antisense, agonist, antagonist, ligand etc. may be labeled.

The conjugation of a saccharide moiety to a biomolecule that is alabeling agent allows the conjugate to be used for detecting thepresence and/or location of particular molecules (e.g., receptors whichbind the saccharide), organelles and/or cell types (e.g., organelles orcell types which bind to and/or accumulate the saccharide). For example,a conjugate comprising a biomolecule labeling agent and M6P may be usedto detect and/or visualize lysosomes, in which M6P-containingbiomolecules typically accumulate, or receptors which bind M6P moieties.

In an exemplary embodiment, the biomolecule is green fluorescent protein(GFP), which is a well-known fluorophore. As exemplified in the Examplessection herein, GFP conjugated to M6P enters cells, allowingvisualization by fluorescent microscope.

The conjugates described herein may be used, for example, to deliverbiomolecules to specific targets bearing a receptor which binds thesaccharide moiety of the conjugate. Thus, a polynucleotide oroligonucleotide biomolecule that is complementary to mRNA may bedelivered to a particular target in order to inhibit expression of aspecific protein via RNA interference, a biomolecule which inhibits aspecific enzyme may be targeted to a cell or organelle (e.g., alysosome) where the enzyme is located, and a biomolecule which is toxicmay be delivered to cancer cells.

The conjugation of the glycosylation reagent and the biomolecule ispreferably effected by reacting the glycosylation reagent describedherein, in which the linker comprises reactive group, with thebiomolecule.

The reactive groups of the linker may be conjugated, for example, todifferent accessible functional groups of the biomolecule.

The phrase “accessible functional group”, as used herein, refers to afunctional group in the surface area of the polypeptide that isaccessible to the molecules of the solvent it is dissolved in. Thesolvent-accessible surface is often referred to as the Lee-Richardsmolecular surface [Lee B. and Richards F M., 1971, “The interpretationof protein structures: estimation of static accessibility”, J. Mol.Biol., 55(3), pp. 379-400]. A functional group of an amino-acid residuewhich is positioned at or near the solvent-accessible surface of aprotein is more likely to be available for chemical modifications andpolymer conjugation, such as PEGylation.

As used herein, the phrase “functional group” describes a chemical groupwhich exhibits a characteristic chemical property, and includes, forexample, the alkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic,halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy,sulfinyl, sulfonyl, cyano, nitro, azide, phosphonyl, phosphinyl, oxo,carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,sulfonamido, and amino chemical groups defined herein. Exemplaryfunctional groups commonly found on biomolecules include, withoutlimitation, alkyl, aryl, heteroaryl, hydroxy, thiol, amine, carboxylate,amide, guanidino, thioalkoxy, phosphate and sulfate.

Thus, for example, the reactive groups of the linker can be conjugatedto functional groups of accessible amino acids on the protein to bemodified, preferably carboxylate (e.g., in glutamate and aspartateresidues), thiol (e.g., in cysteine residues), amine (e.g., in lysineresidues) or hydroxy (e.g., in serine or threonine residues) groups. Inaddition, the linker can be conjugated to functional groups, typicallyhydroxy, of accessible saccharide units of a glycoside (e.g., aglycoprotein).

A functional group may optionally be introduced to a biomolecule bymodification of the biomolecule. For example, thiol groups may beintroduced (i.e., “thiolation”) to the side chains of residues (e.g.,lysine residues) in proteins according to methods known in the art.Aldehyde group may be introduced by oxidizing a hydroxy group,preferably a saccharide-derived hydroxy group as described hereinabove.

As discussed hereinabove, the reactive groups of the linker may beoptionally conjugated to a wide variety of functional groups on aprotein. Hence, the conjugation described herein is not limited tonatural glycosylation sites on proteins, such as specific asparagineresidues (i.e., in N-linked glycosylation) or serine or threonineresidues (i.e., in O-linked glycosylation), and is therefore moreversatile.

Conjugation of the reactive group of the linker to a functional group ofthe biomolecule results in the formation of a covalent bond between thereactive group and the functional group.

In an optional embodiment, the reactive group is a carboxylate, asdescribed herein, and the functional group is an amine. Alternatively,the reactive group is an amine and the functional group is acarboxylate. Typically, conjugation of such groups forms an amide.Preferably, the amine is not a tertiary amine (i.e., preferably at leastone of R′ and R″ is hydrogen). More preferably, the amine is a primaryamine (i.e., R′ and R″ are both hydrogen).

In an alternative embodiment, the reactive group is maleimide and thefunctional group is a thiol. Typically, conjugation of such groupsinvolves addition of the thiol to the C═C bond in the maleimide, forminga succinimide group covalently bound to the sulfur atom of the thiol.

In another example, a reactive group (e.g., carboxy) activated with anNHS group is easily coupled to primary amine groups of a biomolecule,such as a lysine residue on a protein. This coupling is preferablycarried out at slightly alkaline pH levels, ensuring that the amines arenot protonated and can act as good nucleophiles. Alternatively, thereactive group comprises a carboxylate (e.g., carboxylic acid) group andcan be conjugated to a protein's primary amines via the well establishedcarbodiimide coupling chemistry. Similarly, reactive groups comprisingan amine group can be coupled to free carboxylic groups on the protein,as in aspartic or glutamic acids, via the same carbodiimide chemistry.Linker chains comprising a maleimide group can be used for coupling thelinker chain to free thiol groups, such as in cysteines. In general,various bio-conjugation methods can be used to conjugate the reagents ofthe invention to biomolecules.

A biomolecule may be modified with one or more molecules of theglycosylating reagent. Thus, the biomolecule may bound, for example, to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20and even more (e.g., 100) molecules of the glycosylating reagent (onaverage).

The proportion of glycosylation reagents per biomolecules can becontrolled by the conjugation chemistry used. Thus, the identity andnumber of possible amino acids for conjugation may limit the totalnumber of glycosylation reagents that can be attached to each protein,even when an excess of the glycosylation reagent is used.

Thus, the proportion will depend, for example, on the ratio ofglycosylating reagent to biomolecule in the reaction mixture, on thenumber of accessible functional groups capable of being bound by thereactive group of the glycosylating agent, and in cases where thereactive group is activated, on the ratio of glycosylating reagent andan activator thereof (e.g., a carbodiimide reagent and/or NHS). Theproportion can be determined and controlled by a person skilled in theart through routine experimentation.

A biomolecule may be modified with a combination of differentglycosylation reagents to enable binding or interaction with multiplereceptors or to increase the probability of binding to a receptor.

In some cases a target amino acid to be coupled to the glycosylatingreagent is found in the active site of the protein. In such cases,conjugation to the active site amino acid would lead to loss of activityof the protein. For example, a common conjugation methodology toproteins includes targeting the primary amines of lysine residues on thesurface of proteins. Human α-galactosidase-A has 16 lysine residues inits sequence and most of these are accessible to the solvent. However,Lys 168 seems to be located in the active-site itself and evenparticipate in the recognition and catalytic machinery. In such cases,conjugation of the desired glycosylation reagent can optionally takeplace in the presence of an appropriate reversible inhibitor of thespecific enzyme to be modified. Optionally, the inhibitor is found inexcess and the active site of the enzyme is predominantly occupied bythe inhibitor and the active site amino acids are sterically protectedfrom conjugation reagents. The inhibitor is easily evacuated duringdialysis, ultra-filtration or chromatographic steps required to removeexcess conjugation reagents and side-products.

In the case of human α-galactosidase-A, conjugation of an M6Pylationreagent to surface lysines may take place in the presence of inhibitorssuch as 1-deoxynojirimycin.

Following the conjugation, the obtained conjugate (e.g., glycosylatedprotein) can be further purified. Purification is advantageous forremoving excess glycosylation reagents, as well as other reagents usedin the conjugation protocol or side products formed. Purification stepscan also purify or separate modified biomolecules in which themodification profiles are significantly different, for example, proteinswhich were significantly insufficiently modified or alternatively overmodified.

Purification steps employed for the purification of, for example, themodified (conjugated) proteins of the invention include common proteinpurification steps known in the art. These may include dialysis orultra-filtration procedures, which are especially efficient in removinglow molecular weight species, such as excess glycosylation reagents,other coupling reagents or catalysts, as well as organic co-solventsused in the glycosylation step. Dialysis using membranes with a cut-offof about 10,000 are the most efficient at this stage. Other purificationtechniques, such as ammonium sulfate precipitation or acetoneprecipitation may be employed to separate the modified proteins from oneor more contaminant or ingredient described above.

In case further purification steps are required, the partially purifiedconjugated proteins can be further purified by a variety ofchromatography techniques. These may include size-exclusion or gelfiltration chromatography, as well as a variety of ion exchange andhydrophobic chromatographic separation steps. Hydrophobic chromatographyenables the differentiation between proteins with different levels ofmodifications.

The modified proteins (conjugates) of the invention carry uniquepredetermined monosaccharides, according to the glycosylation reagentused. These terminal monosaccharides are useful in affinitychromatography using lectins or lectin-like species, specific for themonosaccharide that is conjugated to the protein. Such affinitychromatography methodology is extremely useful in the separation ofnon-conjugated proteins and conjugated proteins and by differentiatingbetween conjugated proteins according to the level of theirderivatization.

The glycosylation of proteins with the reagents described hereintypically changes some of their biochemical properties. Thus, themodified proteins are optionally analyzed for changes in theirbiochemical properties. Purity and molecular weight of the modifiedprotein may be analyzed by SDS or non-denatured PAGE, followed bystaining with an appropriate visualization dye, such as coomassie blueor silver stain, or using Western blotting. The molecular weight of themodified proteins is more accurately analyzed by mass spectrometrymethods (MALDI-TOF or ESI). Changes to the isoelectric point (pI) of themodified proteins may be ascertained by using isoelectric focusing(IEF). All these methods, as well as other common biochemical proteinanalyses, are well known in the art and some are described in theExamples section that follows. Other common methods can also be applied.

As used herein, the phrase “biochemical properties” refers to the basicchemical properties of a molecule in an environment (e.g., an aqueoussolution at a temperature of 25° C. or 37° C.) characteristic of abiological system. Exemplary biochemical properties include molecularweight, molecular structure, acidity and electrostatic charge.

In one embodiment, when a biomolecule has a biological activity (e.g.,enzymatic activity), the biological activity of the biomolecule isunaffected by its conjugation.

In other embodiments, as detailed hereinbelow, the biological activityof the biomolecule is modified (e.g., enhanced or reduced) upon itsconjugation.

As used herein, the phrase “biological activity” refers to aninteraction with one or more biomolecules and/or other molecules and/orions present in a biological system (e.g., in a cell or in an organism),wherein the interaction produces a biologically significant effect.Examples of biological activities include, without limitation, specificbinding, catalytic activity, cell signaling, immunogenic properties andcell uptake.

Thus, for example, a catalytic activity of an enzyme conjugated to asaccharide may optionally be the same or greater than its catalyticactivity when non-conjugated.

A catalytic activity may optionally be increased by increasing thecatalytic coefficient (k_(cat)) of an enzyme, indicating more efficientconversion of a substrate bound to an enzyme, or optionally by enhancingthe binding of a substrate to the enzyme (e.g., reducing theMichaelis-Menten constant K_(m)).

Optionally, the glycosylating reagent is selected for conjugation to aparticular enzyme such that the saccharide moiety and/or linker bound tothe enzyme increase the catalytic activity thereof. Optimalglycosylation reagents may be selected following routine experimentationwith various glycosylation reagents described herein.

Natural glycosylation as well as protein modifications have been knownto affect the tertiary structure of proteins, hence influencing theaffinity between the protein and its receptor or substrates in the caseof enzymes. Thus, the glycosylation may enable higher affinity towardsthe transition state of the catalyzed reaction, reducing in turn theenergetic barrier of the catalyzed reaction. Alternatively,glycosylations, and other protein modifications, may create amicro-environment for the protein, enabling better interaction with aligand or substrate or stabilizing the protein in a specific environment(e.g. enhancing its solubility).

In cases where the modified biomolecule is an enzyme, the latter isoptionally analyzed to ensure that the conjugation has not causedpartial or full inactivation of their enzymatic machinery. Activityanalysis can be used to fine-tune the modification protocol, level ofmodification, and specific sites of modification.

The Michaelis-Menten kinetic parameters of the modified enzymes may beanalyzed according to known protocols (“Structure and Mechanism inProtein Science”, Alan Fersht, W. H. Freeman and Company, 1999).

For example, the activity of the exemplary enzyme α-galactosidase A iscommonly analyzed by following the enzyme-catalyzed hydrolysis of modelgalactopyranoside substrates in an appropriate activity buffer,preferably in a buffer of relatively low pH, mimicking the pH conditionsin lysosomes, such as 4.6 (Kulkarni et al., Biotechnol. Appl. Biochem.2006, 45, 51; Dean et al. The Journal of Biological Chemistry, 1979, 254(20), 9994). These substrates includep-nitrophenyl-α-D-galactopyranoside and4-methylumbelliferyl-α-D-galactopyranoside. The hydrolysis reactions arecarried out at 37° C. and are terminated by quenching with anappropriate alkaline solution, yielding the desired chromophore ofp-nitrophenol or fluorophore of 4-methylumbelliferol. The level ofhydrolyzed substrates is quantified spectrophotometrically.

The binding affinity of the biomolecule in the conjugate to a target(e.g., a receptor for the biomolecule) may optionally be substantiallyidentical to that of the unmodified biomolecule.

Alternatively, the binding affinity of the conjugate may be greater thanthe binding affinity of the unmodified biomolecule.

A biomolecule may be capable of binding to a plurality of targets, inwhich case it may be characterized by a plurality of binding affinities.The binding affinities of the biomolecule to each of the targets mayindependently be increased, decreased or unaffected by the conjugationof the biomolecule.

In an optional embodiment, the binding of the biomolecule (e.g., ahormone) effects a cell signaling process.

A conjugate may optionally have an increased binding affinity to areceptor, such that a biological activity of the conjugate is greaterthan a corresponding biological activity in an unmodified biomolecule.

Alternatively or additionally, a conjugate may optionally have adecreased binding to a molecule which eliminates the biomolecule (e.g.,by metabolism and/or inactivation), such that the conjugate has a longerlasting biological activity than an unmodified biomolecule.

The binding affinities of a conjugate and the corresponding biomoleculeto a target may be determined according to standard protocols.

For example, a solution containing a predetermined concentration of thebiomolecule (conjugated or unmodified) may be contacted with animmobilized target, such as a target covalently bound to a solid surface(e.g. a bead). The immobilized target is then rinsed, and the quantityof remaining biomolecule is determined by any method known in the art.Repetition of the experiment using different concentrations of thebiomolecule allows the determination of a binding affinity constant, asis well-known in the art. Alternatively, the biomolecule may beimmobilized and contacted with a solution containing the target.

Carbohydrates on the surface of proteins are known to elicit immunogenicresponses. Different methodologies have been devised followingpharmaceutical studies to minimize such response. One of thesemethodologies involves the masking of surface carbohydrates by usinghigh molecular weight PEG chains. As discussed hereinabove, PEG andsimilar molecular chains may be used as a linker in embodiments of thepresent invention. Thus, the linkers in the conjugates of the presentembodiments are optionally selected so as to diminish immunogenicity ofglycosides on the surface of the protein.

The immunogenicity of the modified proteins is optionally analyzed bystandard protocols. In general, a formulation of a modified protein isinjected to mice and the response of their immune system is evaluated bythe total level of immunoglobulins as well as the generation of specificantibodies. The immunogenic response is compared to the response of theunmodified proteins.

The modified proteins bind to receptors with natural affinity towardsthe saccharide used in the modification. This binding may lead tointernalization of the modified protein into target cells, and even intospecific organelles, such as lysosomes.

Uptake of a modified protein can be ascertained by contacting themodified protein with target cells or organelles in vitro and/or invivo, and measuring an amount of the protein in the target cells ororganelles. The amount of a protein may be measured by any suitablemethod used in the art.

In embodiments wherein a biological activity of the biomolecule involvesuptake by a particular cell type (e.g., cells characterized by a lack ofactivity of the biomolecule), the conjugate described herein isoptionally characterized by higher affinity to the cells than thebiomolecule per se.

Optionally, the affinity of the modified and non-modified biomoleculesto the cell type is determined by incubating the biomolecule with aculture of the cell type for a period of 1 to 24 hours (e.g., 5 hours),replacing the cell medium, collecting and lysing the cells, and assayingthe obtained cell extract for the presence of the biomolecule accordingto a standard assay for the biomolecule.

In exemplary embodiments, uptake of a conjugated biomolecule is at least10% higher than uptake of a corresponding non-modified biomolecule.Optionally, the uptake is increased by at least 15%, optionally by atleast 50%, optionally by at least 75%, optionally by at least 100%,optionally by at least 150%, optionally by at least 200%, and optionallyby at least 300%.

For example, proteins may be detected using immunological methods, suchas Western blotting and/or immunohistochemistry, by using an antibodyagainst the protein. Such antibodies may be obtained commercially, or byinducing an immune response in an animal against the protein. Antibodiesmay be labeled (e.g., with a chromophore, fluorophore and/or radiolabel)using techniques known in the art, for use in methods (e.g.,immunohistochemistry) in which labeled antibodies are preferred.

Alternatively, the uptake of an enzyme may be quantified by measuringthe rate of a reaction catalyzed by the enzyme. Optionally, the reactionproduces or consumes a compound which is readily detected (e.g., achromophore or fluorophore).

For in vivo testing, the uptake into different tissue types can becompared by collecting the different tissue types and measuring theuptake in each tissue.

For in vitro testing, the uptake into different cell types may becompared by comparing results obtained using cell cultures of differentcell types.

The enhanced uptake of a modified protein can be ascertained bycomparing uptake of modified and non-modified proteins.

In an exemplary embodiment, the uptake of an α-galactosidase-A modifiedwith M6P-PEG₈-COOH (M6P(α1)—O—(CH₂CH₂O)₈—CH₂CH₂COOH) by fibroblasts of aFabry patient is evaluated. Fabry fibroblasts are pre-cultured in anappropriate medium and incubated with the modified α-galactosidase-A.Following incubation, the cells are washed, centrifuged, lysed and thelevel of up-taken enzyme is analyzed using thep-nitrophenyl-α-D-galactopyranoside activity assay (see the Examplessection below).

In exemplary embodiments, the conjugate comprises M6P-PEG₈-COOH,M6P-PEG₈-maleimide or M6P-PEG₁₂-COOH as described hereinabove,conjugated to a biomolecule. Optionally the biomolecule is anα-galactosidase (e.g., α-galactosidase A), a glucocerebrosidase or GFP.

The above conjugates can be prepared, for example, by conjugating theabovementioned proteins to M6P-PEG₈-COO—NHS(M6P(α1)—O—(CH₂CH₂O)₇—CH₂CH₂COO—NHS.

In another exemplary embodiment, the conjugate has the formula:

wherein B is the biomolecule. This conjugate may be prepared byconjugating the biomolecule with the glycosylating reagent shown in FIG.3A.

As can be seen in the above formula, a maleimide group in aglycosylating reagent typically does not retain the structure ofmaleimide in the conjugate. Rather, the maleimide group reacts so as toproduce a succinimide moiety bound to the biomolecule, typically tosulfur atom of a thiol in the biomolecule. However, the succinimidegroup in a conjugate may be referred to herein as a “maleimide”, inorder to make clear which glycosylating reagent produces the conjugate.

In another exemplary embodiment, the conjugate has the formula:

wherein B is the biomolecule. This conjugate may be prepared byconjugating the biomolecule with the glycosylating reagent shown in FIG.3B.

In another exemplary embodiment, the conjugate has the formula:

wherein B is the biomolecule. This conjugate may be prepared byconjugating the biomolecule with the glycosylating reagent shown in FIG.2A.

In another exemplary embodiment, the conjugate has the formula:

wherein B is the biomolecule. This conjugate may be prepared byconjugating the biomolecule with the glycosylating reagent shown in FIG.2B.

In another exemplary embodiment, the conjugate has the formula:

wherein B is the biomolecule. This conjugate may be prepared byconjugating the biomolecule with the glycosylating reagent shown in FIG.2C.

In another exemplary embodiment, the conjugate has the formula:

wherein B is the biomolecule. This conjugate may be prepared byconjugating the biomolecule with the glycosylating reagent shown in FIG.2D.

The glycosphingolipid ceramide trihexoside (Gb3) accumulates in Fabrydisease due to improper or lack of activity of endogenousα-galactosidase-A. Thus, the levels of Gb3 and its hydrolytic product,lactosylceramide, are optionally evaluated in the Fabry fibroblasts,following their incubation with the M6P-modified α-galactosidase-A.Reduced levels of Gb3 and elevated levels of lactosylceramide are anexcellent indication not just to the mere uptake of the M6P-modifiedα-galactosidase-A but to its catalytic activity within the cells. Washedand lysed Fabry fibroblasts that are incubated with M6P-modifiedα-galactosidase-A are extracted with chloroform:methanol (2:1) andfollowing further RP-18 chromatography, the lipids are analyzed usingthin layer chromatography against Gb3 and lactosylceramide standards.The latter are also used to create a calibration curve which is used toquantify the levels of Gb3 and lactosylceramide following treatment withthe M6P-modified α-galactosidase-A.

The modification with the glycosylation reagents of the invention leadsto uptake of the exogenous proteins and enables exerting theirtherapeutic/biological activity at the target cells by enabling theirbinding to the target receptor. The modification with the glycosylationreagents of the invention can also lead to stabilization of the proteinand enhancement of its half-life in serum and control of itsimmunogenicity. Enhancement of half-life in serum is of extremetherapeutic importance in case the protein is required to react withreceptors and/or be absorbed into target cells in order to exert itsactivity. Enhancement of half-life in serum is also important forbiomolecules which exert an activity in the serum, for example,degrading a specific toxic material. As described hereinabove, thehalf-life in serum of a protein is optionally increased by modifying theprotein with a sialic acid moiety.

The modification of biomolecules with the glycosylation reagentspresented herein therefore leads to a change in the interaction profileof the biomolecule as a function of the saccharide moiety utilized. Thesaccharides which are added to the biomolecule via the glycosylationreagent can result, for example, in interaction (binding) of thebiomolecules with new saccharide binding receptors, in interaction ofthe biomolecule with multiple receptors and/or multiple binding sites,in an improved interaction of the biomolecule with saccharide bindingreceptors (in terms of e.g., higher affinity and/or increased number ofinteraction sites), in reduced immunogenity, and/or in increasedaffinity to target organs, tissues or cells such as macrophages, livercells, spleen cells, kidney cells, epithelial cells, etc.

The conjugates described herein are therefore characterized by featuresthat render these conjugates highly beneficial for use in variousmedical applications, including therapeutic and research applications.

For example, the conjugate may localize the biomolecule therein to aparticular cell type, organelle or receptor, as discussed hereinaboveregarding M6P moieties, and/or the conjugate may enhance the activity ofa biomolecule, as discussed hereinabove regarding sialic acid moieties.

Thus, according to the present embodiments, there is provided apharmaceutical composition that comprises a conjugate as describedherein and a pharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the conjugates described herein, with other chemicalcomponents such as pharmaceutically acceptable and suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare: propylene glycol, saline, emulsions and mixtures of organicsolvents with water, as well as solid (e.g., powdered) and gaseouscarriers.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the conjugates intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the conjugates of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline bufferwith or without organic solvents such as propylene glycol, polyethyleneglycol.

For transmucosal administration, penetrants are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the conjugates of the invention can beformulated readily by combining the conjugates with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable theconjugates of the invention to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for oral ingestion by a patient. Pharmacological preparations for oraluse can be made using a solid excipient, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active conjugate doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theconjugates may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the conjugates for use according tothe present invention are conveniently delivered in the form of anaerosol spray presentation (which typically includes powdered, liquifiedand/or gaseous carriers) from a pressurized pack or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the conjugates and a suitable powder basesuch as, but not limited to, lactose or starch.

The conjugates described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the conjugate preparation in water-soluble form.Additionally, suspensions of the conjugates may be prepared asappropriate oily injection suspensions and emulsions (e.g.,water-in-oil, oil-in-water or water-in-oil in oil emulsions). Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents, which increase the solubility ofthe conjugates to allow for the preparation of highly concentratedsolutions.

Alternatively, the conjugates may be in powder form for constitutionwith a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The conjugates of the present invention may also be formulated in rectalcompositions such as suppositories or retention enemas, using, e.g.,conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.More specifically, a therapeutically effective amount means an amount ofconjugates effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any conjugates used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromactivity assays in animals. For example, a dose can be formulated inanimal models to achieve a circulating concentration range that includesthe IC₅₀ as determined by activity assays (e.g., the concentration ofthe test conjugates, which achieves a half-maximal increase in abiological activity of the conjugate). Such information can be used tomore accurately determine useful doses in humans.

As is demonstrated in the Examples section that follows, atherapeutically effective amount for the conjugates of the presentinvention may range between about 1 μg/kg body weight and about 500mg/kg body weight.

Toxicity and therapeutic efficacy of the conjugates described herein canbe determined by standard pharmaceutical procedures in experimentalanimals, e.g., by determining the EC₅₀, the IC₅₀ and the LD₅₀ (lethaldose causing death in 50% of the tested animals) for a subjectconjugate. The data obtained from these activity assays and animalstudies can be used in formulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain thedesired effects, termed the minimal effective concentration (MEC). TheMEC will vary for each preparation, but can be estimated from in vitrodata; e.g., the concentration necessary to achieve the desired level ofactivity in vitro. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen, which maintainsplasma levels above the MEC for 10-90% of the time, preferable between30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the condition to betreated, dosing can also be a single administration of a slow releasecomposition described hereinabove, with course of treatment lasting fromseveral days to several weeks or until cure is effected or diminution ofthe disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA (the U.S. Food and DrugAdministration) approved kit, which may contain one or more unit dosageforms containing the active ingredient. The pack may, for example,comprise metal or plastic foil, such as, but not limited to a blisterpack or a pressurized container (for inhalation). The pack or dispenserdevice may be accompanied by instructions for administration. The packor dispenser may also be accompanied by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may be of labelingapproved by the U.S. Food and Drug Administration for prescription drugsor of an approved product insert. Compositions comprising a conjugate ofthe invention formulated in a compatible pharmaceutical carrier may alsobe prepared, placed in an appropriate container, and labeled fortreatment of an indicated condition or diagnosis, as is detailed herein.

Thus, according to an embodiment of the present invention, depending onthe selected conjugates, the pharmaceutical composition described hereinis packaged in a packaging material and identified in print, in or onthe packaging material, for use in the treatment of a condition in whichthe activity of the conjugate is beneficial, as described hereinabove.

Further, there is provided a use of the conjugates described herein inthe manufacture of a medicament. The medicament can be for treating avariety of diseases and disorders, depending on the biomolecule utilizedand the nature of the glycosylation reagent. In exemplary embodiments,the medicament can be used in enzyme replacement therapy (ERT), hormonereplacement therapy, as vaccines, and the like.

As used herein, the phrase “enzyme replacement therapy” describes atherapy wherein an enzyme is administered to a patient in whom thatenzyme is deficient or absent. An enzyme may be deficient in quantity(e.g., expressed at lower than normal levels) and/or in activity (e.g.,the activity of the enzyme is reduced or eliminated due to a mutation).

As used herein, the phrase “hormone replacement therapy” describes atherapy wherein a hormone or an analog of a hormone is administered to apatient in whom that hormone is deficient or absent.

As used herein, a hormone or enzyme is termed “deficient” if there is adisease, disorder or discomfort associated with the level of the hormoneor enzyme, and the disease, disorder or discomfort is expected to beameliorated, alleviated or prevented by a higher level of the hormone orenzyme in the patient.

The disease or disorder which the medicament is used to treat may be,for example, a metabolic disease or disorder.

As used herein, the phrase “metabolic disease or disorder” describes adisease or disorder associated with an abnormal form of metabolism. Suchconditions (e.g., lysosomal storage diseases) are often a result of adeficiency or absence of an enzyme, and are thus treated with enzymereplacement therapy.

As used herein and in the art, the phrase “lysosomal storage disease”describes a disease associated with a defective function of lysosomesdue to a deficiency or absence of an enzyme, which results in adeleterious accumulation of one or more materials in the lysosomes.

In general, in cases where the biomolecule is a protein, the conjugatesof the present invention may be used to treat a protein-related diseaseor disorder.

As used herein, the term “protein-related disease” describes a diseaseassociated with an abnormal function of one or more proteins (e.g.,enzymes, hormones, cytokines, receptors etc.). The abnormal function maybe, for example, a deficiency of one or more protein.

A protein-related disease may be treated, for example, by administeringa conjugate comprising a protein that is deficient in the patient.Alternatively, the conjugate comprises a protein that counteracts anundesirable activity of another protein in the patient, or enhances anactivity of another protein which is weaker than desired in the patient.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non-limiting fashion.

Materials and Methods

Proteins:

α-Galactosidase A and glucocerebrosidase were recombinantly producedfrom plant cells as described in International Patent ApplicationPCT/IL2008/000576 and Shaaltiel et al., Plant Biotechnology Journal(2007) 5: 579-590.

Green fluorescent protein (Recombinant EGFP Protein, cat#4999-1000) waspurchased from BioVision Research Products (Mountain View, Calif.).

SDS-PAGE Western Blotting:

SDS-PAGE was effected under standard conditions using 12% SDS-PAGE.Electrophoresis was effected with a Criterion™ cell verticalelectrophoresis apparatus (Bio-Rad Laboratories) with premixedelectrophoresis Tris-Glycine-SDS running buffer (Bio-Rad Laboratories).12% acrylamide gels were prepared using premixed solutions of 40%acrylamide/Bis and 10% SDS solution. Transfer of proteins frombis-acrylamide gels to nitrocellulose membrane was effected using aBIO-RAD Criterion™ blotter system overnight at room temperature in 30 V.The membrane was then blocked with PBS containing 5% non-fat dry milk,washed with PBS containing 0.1% Tween-20, and bound to the primary andsecondary antibody using PBS containing 0.1% Tween-20. The primaryantibody used was Rb-anti-α-GalA (1:1500; H-104 sc-25823, Santa Cruz) or1:5000 anti-GCD (prepared as described herein). Detection was preformedwith an ECL detection kit (Pierce). Bands were detected using theMolecular Imager Gel Doc XR System (Bio-Rad Laboratories) for 30″, 60″,90″ as needed.

Mass Spectrometry:

Mass-Spectrometry (MS) analysis was performed using a matrix-assistedlaser desorption ionization time-of-flight/time-of-flight (MALDI-TOF)mass spectrometer (4700, Applied Biosystems, Foster City Calif.) and anion-trap mass spectrometer (LCQ classic, Finnigan, San Jose, Calif.).

α-Galactosidase-A-deficient mice:

Jackson B6J129Gla α-galactosidase-A-deficient mice (“Fabry mice”) werepurchased from Jackson Laboratories. These mice are characterized bybeing totally deficient in α-Galactosidase-A activity and progressivelyaccumulate Gb3 in both plasma and in the lysosomes of most tissues (inparticular, the liver, spleen, heart, skin, and kidneys). In addition,these mice have no clinical disease phenotype and survive a normallaboratory life span (>2 years). Hemizygous affected males were bred tohomozygous affected females, thereby providing only affected offspring.For these studies, all mice were affected adult males 12 to 30 weeks ofage at study initiation.

Fabry Fibroblast Cultures:

Human Fabry (α-galactosidase-deficient) fibroblasts originating fromFabry patients (Cat. ID GM02775, Cornell Institute) were used. Thefibroblasts were cultured in 24-well culture plates containing DMEM(Dulbecco's modified Eagle medium) (cat. D5546, Sigma) supplemented with12% FBS (fetal bovine serum), 5 ml L-glutamine, 5 ml MEM Eagle vitaminsolution, 10 ml MEM amino acid solution, 5 ml MEM Eagle non-essentialamino acid solution and 5 ml Pen-Strep solution, all supplements fromBiological Industries (Beit Haemek, Ill.).

α-Galactosidase-A Assay:

The level of active α-galactosidase A was determined against acalibration curve of the activity of a commercial α-galactosidase(Fabrazyme®, Genzyme, Cambridge, Mass.) plotted for the concentrationrange of 200-12.5 ng/ml. Activity was determined usingp-nitrophenyl-α-D-galactopyranoside (Sigma) as a hydrolysis substrate.The assay buffer contained 20 mM citric acid, 30 mM sodium phosphate,0.1% BSA and 0.67% ethanol at pH 4.6. The assay was performed in 96 wellELISA plates (Greiner #655061). 50 μl of tissue sample lysates wereincubated with 150 μl assay buffer and 30 μl substrate was added toobtain a final concentration of 8 mM. The reaction mixture was incubatedat 37° C. for 90 minutes and results were plotted against thecalibration results. Product (p-nitrophenyl; pNP) formation was detectedby absorbance at 405 nm. Absorbance at 405 nm was measured beforeinitiating the reaction. After 90 minutes, 100 μl of 1.98 M sodiumcarbonate was added to each well in order to terminate the reaction, andabsorbance at 405 nm was measured again.

Anti-GCD Antibodies:

75 μg of recombinant GCD (Cerezyme™) was suspended in 3 ml completeFreund's adjuvant and injected to each of two rabbits. Each rabbit wasgiven a booster injection after two weeks. The rabbits were bledapproximately 10 days after the booster injection and again at one weekintervals until the antibody titer began to drop. Following removal ofthe clot the serum was divided into aliquots and stored at −20° C.

Example 1 Synthesis of Mannose (α1)-O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COO—NHS

As mentioned above, the glycosylation reagents of the invention can besynthesized using a variety of synthetic methodologies. One of thesemethodologies is set forth herein in the synthesis of a specificmannose-containing derivatization reagent (glycosylation reagent):

A protected PEG linker (HO—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOR) issynthesized by mono-protecting the nonaethylene glycol(HO—CH₂CH₂—(OCH₂CH₂)₈—OH) with TBDMS-Cl. The free hydroxyl group isconverted to a chloride by reacting the mono-protected PEG with thionylchloride, using pyridine as a proton scavenger under elevatedtemperature (60° C.). The chloride is further converted to a carboxylgroup by the reaction with Mg° in anhydrous ether and further reactingthe resulting organometallic derivative(TBDMS-O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂MgCl) with carbon dioxide. Followingacidification with H₂SO₄, the carboxylic acid(TBDMS-O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOH) is esterified with methanol toyield the corresponding ester(TBDMS-O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOCH₃). The t-butyldimethylsilylprotecting group is removed by treatment with tetrabutylammoniumfluoride in THF to yield the mono-protected PEG linker,HO—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOR wherein R═CH₃.

A mixture of 1,2,3,4-tetra-O-acetyl-D-mannose, the mono-protected PEGlinker (HO—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOR wherein R═CH₃) and ZnCl₂ isheated at 100-110° C. with stirring under reduced pressure, with a sodalime trap interposed between the reaction vessel and the vacuum sourceto remove acetic acid, for 4 hours. The resulting mass is dissolved inethyl acetate, washed twice with water and dried over anhydrous Na₂SO₄.The ethyl acetate is removed under reduced pressure and the residue ischromatographed on a silica gel 60 chromatography column to give the1-PEG-2,3,4-tri-O-acetyl-D-mannoside. The product is furtherchromatographed to separate the α and β anomers. The α anomer isdeprotected by treatment with 2N sodium methoxide. The resulting solidis filtered and washed with ethyl alcohol to give the corresponding saltsodium salt (Mannose-(α1)—O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COONa).

The glycosylating reagent can be conjugated to a target protein by usinga carbodiimide agent, such as DCC (dicyclohexylcarbodiimide).Alternatively, the carboxylic acid can be activated by transforming itto an N-hydroxy succinimide ester (NHS) or sulfo-NHS ester by reactingthe carboxylic acid with the N-hydroxy succinimide in the presence of anappropriate carbodiimide coupling reagent, such as EDC(1-ethyl-3-(3-diaminomethylpropyl)carbodiimide).

Example 2 Synthesis of M6P(α1)-O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOH

The following describes another exemplary methodology for the synthesisof a specific mannose-containing derivatization (glycosylation) reagenthaving a carboxylate functional group.

A protected PEG linker (HO—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOR) issynthesized as described in Example 1.

A mixture of 1,2,3,4-tetra-O-acetyl-D-mannose, the mono-protected PEGlinker (HO—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOR wherein R═CH₃) and ZnCl₂ isheated at 100-110° C. with stirring under reduced pressure, with a sodalime trap interposed between the reaction vessel and the vacuum sourceto remove acetic acid, for 4 hours. The resulting mass is dissolved inethyl acetate, washed twice with water and dried over anhydrous Na₂SO₄.The ethyl acetate is removed under reduced pressure and the residue ischromatographed on a silica gel 60 chromatography column to give the1-PEG-2,3,4-tri-O-acetyl-D-mannoside. The product is furtherchromatographed to separate the α and β anomers.

To a solution of2,3,4-tri-O-acetyl-mannose-(α1)—O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOCH₃ indry pyridine is added diphenyl chlorophosphate dropwise at roomtemperature over 1 hour and the mixture is allowed to stand at roomtemperature overnight. The mixture is the heated at 40° C. for 3 hours.The solvent is evaporated under reduced pressure and the residuechromatographed on a silica gel chromatography column (silica gel 60) togive2,3,4-tri-O-acetyl-6-diphenylphosphate-mannose-(α1)-β-CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOCH₃.

The latter is dissolved in dry methanol and is hydrogenated in thepresence of platinum oxide catalyst at slightly greater than atmosphericpressure. When the calculated amount of hydrogen is taken up, thecatalyst is removed by filtration and the solvent evaporated underreduced pressure. The residue is chromatographed on a silica gelchromatography column (silica gel 60) to give2,3,4-tri-O-acetyl-M6P-(α1)-β-CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOCH₃, which isfurther deprotected by treatment with 2N sodium methoxide. The resultingsolid is filtered and washed with ethyl alcohol to give the tri-sodiumsalt (M6P(α1)—O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COONa).

Following are the spectral data of(M6P(α1)—O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOH):

¹H NMR (400 MHz, D₂O): δ=4.72 (m, 1H), 3.82 (m, 1H), 3.75 (m, 1H),3.71-3.65 (m, 1H), 3.65 (t, J=6.1 Hz, 2H), 3.61-3.57 (m, 2H), 3.55 (m,32H), 3.5 (m, 1H), 2.52 (t, J=6.1 Hz, 2H) ppm;

¹³C-NMR (D₂O): δ=176.75, 100.21, 71.88, 71.79, 70.50, 70.09, 69.73,69.65, 66.62, 66.50, 66.34, 64.15, 34.81 ppm;

³¹P-NMR (D₂O): δ=1.69 (s);

MS (ESI): 603.4 (M-PO₃H₂).

Example 3 Characterization of Glycosylating Reagents

The glycosylating reagents M6P-PEG₁₂-COOH and M6P-PEG₈-maleimide wereprepared using common methodologies.

Following are the spectral data of M6P-PEG₁₂-COOH:

¹H NMR (400 MHz, D₂O): δ=4.90 (m, 1H), 4.12 (m, 1H), 3.98 (m, 1H),3.94-3.88 (m, 1H), 3.87-3.83 (m, 1H), 3.81 (t, J=6.1 Hz, 2H), 3.78-3.74(m, 2H), 3.73-3.68 (m, 48H), 2.67 (t, J=6.1 Hz, 2H) ppm;

¹³C-NMR (D₂O): δ=176.55, 100.22, 71.87, 71.80, 70.51, 70.09, 69.73,69.66, 69.61, 66.62, 66.42, 66.34, 64.10, 34.65 ppm; and

³¹P-NMR (D₂O): δ=1.64 (s).

Following are the spectral data of M6P-PEG₈-maleimide:

¹H NMR (400 MHz, D₂O): δ=6.86 (m, 1H), 4.90 (m, 1H), 3.75 (m, 1H), 4.12(m, 1H), 3.98 (m, 1H), 3.92-3.83 (m, 1H), 3.76 (m, 2H), 3.74-3.67 (m,28H), 3.62 (t, J=5.2 Hz, 2H), 3.55 (t, J=6.4, 2H), 3.39 (s, 1H), 3.36(t, J=5.2 Hz, 2H), 2.28 (t, J=7.6 Hz, 2H), 1.91 (quintet, J=7.2 Hz, 2H)ppm; and

³¹P-NMR (D₂O): δ=1.64 (s).

Example 4 M6P Derivatization of α-galactosidase-A amines

One methodology for conjugating moieties to human α-galactosidase-A isby targeting the primary amines of the side chains of lysine residues onthe surface of the protein. Human α-galactosidase-A has 16 Lysineresidues in its sequence, most of which are solvent accessible.

The following describes an exemplary procedure according to thismethodology.

1 mg of M6P(α1)-O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COONa, prepared as describedin Example 2 hereinabove, was activated by reaction with EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and S—NHS(sulfo-N-hydroxysucinimide) in a 1:1.3:1.3 molar ratio. The reaction wasperformed in 37.4 μl DMSO (dimethyl sulfoxide), overnight, at roomtemperature. The obtained reaction mixture was used withoutpurification.

The obtained reaction mixture was added to 800 μl of a solution ofa-galactosidase A (1.65 mg/ml) in a phosphate buffer solution (pH 7.4,50 mM). The solution was mixed well and the reaction was then allowed toproceed overnight at 4° C. The modified protein was separated fromlow-molecular weight moieties by dialysis (10,000 Da cut-off) againstphosphate buffer (pH 6, 25 mM).

As shown in FIG. 4, conjugation of M6P-PEG₈-CO₂H to α-galactosidase-Aresulted in an increase in the mass of the α-galactosidase-A, asmeasured by SDS-PAGE.

As shown in FIG. 5, conjugation of M6P-PEG₈-CO₂H to α-galactosidase-Aresulted in a shift of the pI of the α-galactosidase-A.

These results presented in FIGS. 4 and 5 confirm the successfulconjugation of M6P-PEG₈-CO₂H to the protein.

Conjugation of M6P-PEG₈-CO₂H to the protein was performed on a largerscale using the methodology described above.

4.1 mg of S—NHS, 10 mg of M6P(α1)—O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COONa (in200 μl of DMSO) and 4.3 mg of EDC (in 93 μl DMSO) were mixed, and thereaction mixture was stored overnight at room temperature.

263 μl of the obtained reaction mixture was added to 7.2 ml phosphatebuffer solution (50 mM, pH=7.1) containing 1.65 mg/ml α-galactosidase-A.The obtained reaction mixture was stirred at 4° C. overnight. Themodified protein was separated from low-molecular weight moieties bydialysis (10,000 Da cutoff) against phosphate buffer (pH 6, 25 mM).

As shown in FIG. 6, conjugation of M6P-PEG₈-CO₂H to α-galactosidase-Aresulted in a shift of the pI of the α-galactosidase-A.

As shown in FIG. 7, conjugation of M6P-PEG₈-CO₂H to α-galactosidase-Aresulted in an increase in the mass of the α-galactosidase-A, asmeasured by SDS-PAGE.

As shown in FIG. 8, the MALDI-TOF mass spectrum of homo-dimerica-galactosidase-A conjugated to M6P-PEG₈-CO₂H exhibits an increase of5.1 kDa in relation to the mass spectrum of the native homo-dimericα-galactosidase-A, indicating an average incorporation of 7-8M6P-PEG₈-CO₂H moieties per homo-dimer of a-galactosidase-A.

Example 5 M6P Derivatization of Glucocerebrosidase (GCD)

13 mg of M6P-PEG₈-COOH in 260 ml DMSO was added to 4.7 mg of S—NHS. 80μl of freshly prepared EDC solution (5.5 mg in 110 μl DMSO) was thenadded. The obtained reaction mixture was shaken overnight at roomtemperature.

The reaction mixture was then added to 12 mg GCD in 9 ml of phosphatebuffer (50 mM, pH 7.4). The reaction mixture was shaken for 2 hours atroom temperature. The solution was then dialyzed using Vivaspin (6 ml,cutoff of 10,000 Da) against saline.

As shown in FIG. 9, conjugation of M6P-PEG₈-CO₂H to GCD resulted in ashift of the pI of the GCD.

As shown in FIG. 10, conjugation of M6P-PEG₈-CO₂H to GCD resulted in anincrease in the mass of the GCD, as measured by SDS-PAGE.

As shown in FIGS. 11A-B, the MALDI-TOF mass spectrum of GCD conjugatedto M6P-PEG₈-CO₂H exhibits an increase of 2.5 kDa in relation to the massspectrum of the native GCD, indicating an average incorporation of 3-4M6P-PEG₈-CO₂H moieties per GCD molecule.

These results presented in FIGS. 9-11 confirm the successful conjugationof M6P-PEG₈-CO₂H to GCD.

Example 6 Preparation of M6P-Derivatized GFP

GFP (Green fluorescent protein) was modified by M6P-PEG₈-COOH, using themethodology described in Example 3, to test uptake of a modified proteinvia M6P receptor.

M6P-PEG₈-COOH was activated by EDC and S—NHS in a 1:1.1:1.1 ratio(M6P-PEG₈-COOH:EDC:S—NHS) in DMSO. After the activation, GFP was addedin phosphate buffer (66 mM, pH 8) and the reaction mixture was storedovernight at 4° C. The results were analyzed by SDS-PAGE (12%),isoelectric focusing and mass spectroscopy.

As shown in FIG. 12A, conjugation of M6P-PEG₈-CO₂H to GFP resulted in anincrease in the mass of the GFP, as measured by SDS-PAGE.

As shown in FIG. 12B, conjugation of M6P-PEG₈-CO₂H to GFP resulted in ashift of the pI of the GFP.

As shown in FIGS. 13A-B, conjugation of M6P-PEG₈-CO₂H to GFP resulted inan increase in the mass of the GFP, as measured by mass spectrometry.

The above results confirm that M6P-PEG₈-CO₂H was successfully conjugatedto GFP.

Example 7 M6P Derivatization of α-Galactosidase-A Through Protein Thiols

Maleimide-activated reagents are effective for protein modification ofsulfhydryl groups. Maleimide groups react efficiently and specificallywith free (reduced) sulfhydryls at pH 6.5-7.5 to form stable thioetherbonds. Thus, a suitable reagent for M6P derivatization ofα-galactosidase-A through protein thiols is a maleimide activatedM6P-PEG.

Protein modification via free sulfhydryl (thiol) groups is effected viaconjugation to free thiol moieties of the side chain of cysteineresidues. Human α-galactosidase-A has 12 cysteine residues, 10 of whichare engaged in intra-molecular disulfide bonds and cannot be targeted bymaleimide activated M6P-PEGs. One of the free cysteine residues (Cys 90)is buried in the protein and is not accessible for conjugation. However,Cys 174 is relatively exposed to the solvent and can be used formono-derivatization of human α-galactosidase-A.

As human α-galactosidase A has only one cysteine residue available forconjugation, the lysine residues of α-galactosidase A were thiolatedusing Traut reagent (2-iminothiolane hydrochloride) and then coupled toa M6Pylation reagent terminated with the maleimide reactive group.

A stock solution of 10 mg/ml Traut's reagent in DMSO was prepared justbefore the reaction. 80 μl of the Traut's stock solution was added to1.6 ml of α-galactosidase A (3.2 mg/ml) that was diluted with 1.6 ml ofphosphate buffer (pH 8, 50 mM). Following incubation for 30 minutes atroom temperature, 4 ml of phosphate buffer (pH 6, 200 mM) was added. Theobtained reaction mixture was dialyzed via Vivaspin 6 (Sartorius) with a50 kDa cutoff in to phosphate buffer (pH 6 50 mM).

1 mg of M6P-PEG₈-maleimide (as depicted in FIG. 1B) in 20 μl DMSO wasadded to the reaction mixture, and the reaction mixture was then shakenfor 8 hours at 4° C. An additional 1 mg of M6P-PEG₈-maleimide was thenadded. After being kept for two days at 4° C., the obtained reactionmixture was purified using dialysis via Vivaspin with a 50 kDa cutoff insaline solution.

The derivatized protein was further analyzed by SDS-PAGE and isoelectricfocusing.

As shown in FIG. 14A, conjugation of M6P-PEG₈-maleimide toa-galactosidase-A resulted in a shift of the pI of theα-galactosidase-A.

As shown in FIG. 14B, conjugation of M6P-PEG₈-maleimide toa-galactosidase-A resulted in an increase in the mass of theα-galactosidase-A, as measured by SDS-PAGE.

These changes in pI and SDS-PAGE migration correspond to an averageincorporation of 1-2 M6P-PEG₈-Mal moieties per α-galactosidase-A dimeras indicated in the MALDI-TOF MS results (not shown).

Example 8 M6P Derivatization of α-Galactosidase-A Through ProteinCarboxylates

Human α-galactosidase-A has 44 carboxylic acid-containing side chains inits sequence, being residues of aspartic and glutamic acids. An M6P-PEGreagent according to the present embodiments can be coupled to thesolvent-exposed carboxylic acid residues by utilizing an amineterminated M6P-PEG derivative, and a carbodiimide catalyst. In order toavoid cross-linking between α-galactosidase-A amines and carboxylicresidues, a large excess of the M6P-PEG reagent is used.

Following is an exemplary synthesis for conjugating amine-terminatedM6P-PEGs to human α-galactosidase-A via protein carboxylates.

α-Galactosidase-A (1 mg/mL) in an appropriate buffer (pH 6) is added toa solution containing the amine terminated M6P-PEG reagent in 50-foldmolar excess, DCC (in 60-fold molar excess) and DMAP (in 100-fold molarexcess) at 4° C. The reaction mixture is kept at 4° C. with gentleagitation for 12 hours and is further dialyzed to remove excess reagentsand by products. The derivatized protein is analyzed by SDS-PAGE.

The reaction can optionally be performed in the presence1-deoxynojirimycin, or any other α-galactosidase-A inhibitor, in orderto avoid loss of enzymatic activity due to conjugation of the M6P-PEGreagent to the aspartic acid residues within the active site.

Example 9 Mannose Derivatization of Proteins

A glycosylating reagent comprising mannose (obtained, for example, asdescribed in Example 2 hereinabove, wherein the phosphorylation of themannose moiety is omitted) can be conjugated to a protein's primaryamine groups by using a carbodiimide agent, such as DCC or EDC,optionally catalyzed by a proton scavenger such as dimethylaminopyridine (DMAP). As described in Example 4, the terminal carboxylic acidcan be activated by transforming it to an NHS or sulfo-NHS ester by thereaction of the carboxylic acid with N-hydroxy succinimide in thepresence of an appropriate carbodiimide coupling reagent, such as EDC.

Following is an exemplary methodology for conjugating a mannose-PEG-COOHreagent to a protein.

Glycosylating Reagent Activation:

A 10-fold molar excess of EDC is added to a glycosylating reagentmannose(α1)—O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COOH, which is preparedaccording to the methodology described in Example 2, to give a 2 mMsolution in a 0.1 M phosphate citrate buffer, pH 5.0. NHS is furtheradded in a 25-fold molar excess. The reaction components are allowed toreact with agitation for 15 minutes at room temperature. The pH iselevated to 6.0 and human GCD is added to the solution to give a 5-foldmolar excess of mannose-PEG-NHS. The solution is mixed well and thereaction is allowed to proceed overnight at 4° C. The modified proteinis separated from low-molecular weight moieties by dialysis (10,000 Dacut-off).

As with α-galactosidase-A, other proteins can typically be derivatizedat free and accessible thiol groups of cysteine residues and carboxylicacid groups of aspartic acid and glutamic acid residues.

Example 10 Activity of Derivatized α-galactosidase-A

A M6P-derivatized α-galactosidase-A, as described, for example, inExample 4 hereinabove, is assayed for its catalytic activity followingits M6Pyaltion, in order to ensure that the derivatization with M6P didnot impair the catalytic activity of the enzyme.

Non-modified α-galactosidase-A is assayed in order to provide a control.

The modified or non-modified enzyme is dialyzed (×2) against 2 Liters ofcitrate phosphate buffer (20 mM, pH 4.6), diluted to give a 50 nMsolution, and is allowed to react at 37° C. with its substrate analog,p-nitrophenyl-α-D-galactopyranoside (5000 μM). The reaction is allowedto continue for 15 minutes and is quenched with 5N NaOH. Theconcentration of the hydrolysis reaction, p-nitrophenol, is measuredspectrophotometrically (401 nm) in a 1 cm cuvette and quantified using ap-nitrophenol calibration curve. The α-galactosidase-A activity isdefined as the μM of p-nitrophenol liberated per 1 minute per 1 nM ofenzyme.

The activity of the M6P-derivatized α-galactosidase-A prepared asdescribed in Example 4 was tested as described herein. Theα-galactosidase-A was found to have retained enzymatic activityfollowing derivatization.

Example 11 Activity of Mannose Derivatized GCD

A derivatized glucocerebrosidase as described in Example 9 hereinaboveis assayed for its catalytic activity following its mannosederivatization, so as to ensure that the derivatization did not impairthe catalytic activity of the enzyme.

Following is an exemplary methodology for assaying the catalyticactivity of mannose derivatized GCD. The enzyme is dialyzed (×2) against2 liters of phosphate citrate buffer (0.1M, pH 5.5), diluted to give a25 nM solution and allowed to react at 37° C. with its substrate analog,p-nitrophenyl-beta-D-glucopyranoside (5000 μM). The reaction is allowedto continue for 15 minutes and is then quenched with 5 N NaOH. Theconcentration of the hydrolysis product, p-nitrophenol, is measuredspectrophotometrically (401 nm) in a 1 cm cuvette and quantified using ap-nitrophenol calibration curve. The GCD activity is defined as the μMof p-nitrophenol liberated per 1 minute per 1 nM of enzyme.

Example 12 Biochemical Properties of M6P and Mannose Derivatized GCD

A derivatized glucocerebrosidase as described in Example 9 hereinaboveis assayed for its biochemical properties following its mannose or M6Pderivatization, so as to ensure that the derivatization with mannose orM6P residues does not impair the catalytic characteristics of theenzyme, and to establish its new biochemical properties, including itsmolecular weight (Mw), isoelectric point (pI), and its Michaelis-Mentenconstant (K_(M)) as well as its maximum velocity (V_(max)) and catalyticcoefficient (k_(cat)). Furthermore, human GCD is a monomer and furtheranalyses are carried out to ensure that the enzyme retains its monomericform following mannose derivatization.

The Mw of the derivatized GCD is assayed by mass spectrometry(MALDI-TOF). The existence of the monomeric form can also be establishedby a gel-filtration analysis using an analytical HPLC. TheMichaelis-Menten kinetics are carried out at an enzyme concentration of30 nM using p-nitrophenyl-beta-D-glucopyranoside as substrate, atsubstrate concentrations ranging from 50 μM to 10000 μM. Reactionmixtures are incubated at 37° C. for 10 to 180 minutes, ensuringconversion levels do not exceed 5%. Reactions are then quenched with 5NNaOH and the level of the p-nitrophenol product is quantifiedspectrophotometrically using a calibration curve (401 nm). The velocityof each reaction is calculated by dividing the concentration ofp-nitrophenol by the reaction time and the velocity vs. substrateconcentration curve (Michaelis-Menten plot) is used to calculate theenzyme's K_(M), V_(max) and k_(cat), using non-linear regression.

Non-modified GCD is assayed as described above in order to provide acomparison.

Example 13 In-Vitro Uptake of Derivatized α-Galactosidase-A

Targeting and uptake of α-galactosidase-A to target cells and tissues ismediated by the M6P receptor and can be determined using a fibroblastcell line expressing M6P receptors.

The uptake of α-galactosidase A conjugated with either M6P-PEG₁₂-CO₂H(see FIG. 1A) or M6P-PEG₈-maleimide (see FIG. 1B) was compared with thatof non-modified α-galactosidase A.

Fibroblasts (GM02775, Coriell institute) of Fabry affected Caucasianmale were pre-cultured in 48-well plates (1×10⁵ cells per ml) with DMEM(D5546, Sigma) containing 12% FBS, 200 mM L-Glutamine, MEM Vitmainssolution (01-326-1, Biological industries Ltd., Israel), essential andnon-essential amino acid solutions (Biological industries Ltd., Israel,01-325-1 and 01-340-1, respectively) and Penicillin-Streptomycinsolution, 10,000 units per ml Penicillin G. α-Galactosidase-A wasdiluted in medium, and 50 μl of the solution were then added to eachwell to obtain a final concentration of 0.5-30 μg/ml. Followingincubation of 5 hours, the medium was collected. Cells were washed withPBS and trypsinized. Following neutralization, cells were centrifuged at2,000 g for 4 minutes. Pellets were washed in PBS and centrifuged again.Pellets were vigorously pipetted with 60 μl of lysis buffer containingprotease inhibitors. Lysed cells were frozen and thawed twice andα-galactosidase A uptake was measured by Western blotting, as describedhereinabove.

As shown in FIGS. 15A-B, uptake of α-galactosidase A into Fabryfibroblasts was increased following conjugation with eitherM6P-PEG₁₂-CO₂H or M6P-PEG₈-maleimide.

Example 14 In-Vivo Uptake of α-Galactosidase-A Derivatized withM6P-PEG₈-CO₂H

Targeting and uptake of α-galactosidase-A to target organs and tissuesis mediated by the M6P receptor and was determined by measuring thelevels of alpha-galactosidase-A in the tissues of “Fabry Mice” (JacksonB6J129Gla). α-Galactosidase-A accumulation in mouse liver and spleenfollowing i.v. administration of derivatized α-galactosidase-A anduntreated α-galactosidase-A is measured by enzymatic activity.

Fabry mice received a single intravenous (i.v.) bolus injection ofa-galactosidase-A or derivatized α-galactosidase-A (conjugated toM6P(α1)—O—CH₂CH₂—(OCH₂CH₂)₇—O—CH₂CH₂COONa, as described in Example 4hereinabove, and referred to herein as M6P-PEG₈-CONH-α-galactosidase-A).Prior to injection, each sample for injection was tested for proteinactivity as described hereinbelow. Animals were sacrificed 24 hours or 7days post-injection. The experiments performed are summarized in Table 1below.

TABLE 1 in vivo M6P-PEG₈-CONH-α-Gal-A experiments Number Dosing 24 7 ofvolume Group hours days animals (ml/kg) Comments α-Gal-A 3 3 6 mice 5Test group 18.75 mg/kg M6P-PEG₈₋ 4 4 8 mice Test group CONH-α- Gal-A 3mg/kg M6P-PEG₈₋ 4 4 8 mice Test group CONH-α- Gal-A 10 mg/kg Saline 3 36 mice Negative control

Prior to sacrifice (on Day 1 or 7) individual blood samples wereobtained by retro-orbital sinus bleeding under anesthesia. The volume ofblood obtained did not exceed 15% of the circulating blood volume (72ml/kg). Samples were collected to pre-labeled Li-Heparinized coatedtubes (mini collect 0.5 mL tubes, Greiner Bio One, Cat#6-450479), andcentrifuged to obtain plasma samples.

Following blood collection, animals were perfused (to remove heme, whichinterferes with the fluorometric a-Gal A enzymatic assay) with 0.9%saline, administered to the left ventricle of the heart, with concurrentsevering of the right atrium or jugular. Perfusion was performed untilno blood poured from the right atrium or jugular, after which the lungs,liver, spleen, heart and kidneys were collected from each mouse. Eachsample was frozen immediately in liquid nitrogen and then transferred tostorage at about −70° C. for enzyme or Gb-3 analyses.

Soluble tissue samples were assayed for α-galactosidase-A activity asdescribed herein 24 hours after injection.

As shown in FIGS. 16A-B and FIG. 17, uptake ofM6P-PEG₈-CONH-α-galactosidase A into the liver was greater than uptakeof plant recombinant α-galactosidase-A.

As shown in FIGS. 18A-B, uptake of M6P-PEG₈-CONH-α-galactosidase A intothe spleen was greater than uptake of plant recombinantα-galactosidase-A.

As shown in FIGS. 19A-B, uptake of M6P-PEG₈-CONH-α-galactosidase A intothe heart was greater than uptake of plant recombinantα-galactosidase-A.

As shown in FIGS. 20A-B, uptake of M6P-PEG₈-CONH-α-galactosidase A intothe lungs was greater than uptake of plant recombinantα-galactosidase-A.

As shown in FIGS. 21A-B, uptake of M6P-PEG₈-CONH-α-galactosidase A intothe kidneys was greater than uptake of plant recombinantα-galactosidase-A.

Soluble tissue samples were also assayed for α-galactosidase-A activity7 days after injection.

As shown in FIGS. 22A-B, 23 and 24, uptake ofM6P-PEG₈-CONH-α-galactosidase A remained higher than uptake of plantrecombinant α-galactosidase A in spleen, liver and heart tissues 7 daysafter injection.

In kidney and lung tissue, no α-galactosidase activity was detectedabove background levels for either modified or unmodifiedα-galactosidase A.

In addition, plasma samples were assayed for α-galactosidase A activity.High plasma levels indicate lack of uptake into the cells.

As shown in FIG. 25, unmodified α-galactosidase A remained in the plasmaboth 24 hours and 7 days after injection, whereas,M6P-PEG₈-CONH-α-galactosidase A was absent from plasma.

The results presented hereinabove demonstrate that modification of plantrecombinant α-galactosidase A with an M6P-PEG₈-COOH moiety considerablyimproved uptake of the α-galactosidase A in a wide variety of tissuetypes both 24 hours and 7 days after injection.

Example 15 In-Vivo Uptake of Additional Forms of Derivatizedα-Galactosidase-A

α-Galactosidase A was derivatized with the glycosylation reagentsM6P-PEG₈-maleimide (depicted in FIG. 1B) and M6P-PEG₁₂-CO₂H (depicted inFIG. 1A). The resulting conjugates are referred to herein asM6P-PEG₈-maleimide-α-galactosidase-A andM6P-PEG₁₂-CONH-α-galactosidase-A, respectively.

Uptake of the derivatized α-galactosidase A was determined in vivo,using the methodology described in Example 14.

Preliminary results were obtained and are shown in FIGS. 26A-B.

As shown in FIGS. 26A-B, the uptake of the derivatized α-galactosidase Ainto heart (FIG. 26A) and lung (FIG. 26B) tissue was greater in mostgroups tested than uptake of non-derivatized α-galactosidase A.

Determination of optimal conditions for conjugation with theaforementioned glycosylation reagents is currently ongoing, in order toallow for more consistent results.

Example 16 In-Vitro Uptake of Mannose Derivatized GCD

Targeting and uptake of GCD to macrophages is mediated by themannose/N-acetylglucosamine (Man/GlcNAc) receptor and can be determinedusing murine thioglycolate-elicited peritoneal macrophages or macrophagecell line expressing Man/GlcNAc receptors.

Following is an exemplary methodology for assaying the in-vitro uptakeof mannose derivatized GCD. Rat macrophage cell line (ATCC#R8383) iscultured in DMEM (Beit Haemek, Israel) containing 10% fetal calf serum,plated at 2-5×10⁵ cells/well in 96-well plates, and incubated at 37° C.for 90 minutes with culture medium containing GCD. Medium issubsequently removed, cells are washed three times with PBS, andactivity is measured in cell extract.

The activity of GCD taken up by the cells is determined by enzymaticactivity assay using p-nitrophenyl-beta-D-glucopyranoside. The assaybuffer contains 4 mM p-nitrophenyl-beta-D-glucopyranoside, 1.3 mM EDTA,0.15% Triton X-100, 0.125% sodium taurocholate, 60 mM phosphate-citratebuffer, pH 6.0. After 60 minutes at 37° C., the reaction is terminatedusing 5N NaOH, and the reaction product (p-nitrophenol) is determined byits absorbance at 405 nm.

Example 17 In-Vivo Uptake of Mannose Derivatized GCD

Targeting and uptake of GCD to macrophages in spleen and liver ismediated by the mannose/N-acetylglucosamine (Man/GlcNAc) receptor andcan be determined by measuring the levels of GCD in the tissues, and thelevels of glucosylceramide, the substrate of GCD. GCD accumulation inmouse liver and spleen following i.v. administration of derivatized GCDand untreated GCD is measured by enzymatic activity and analysis ofglucosylceramide levels. Following is an exemplary methodology forassaying the in-vivo uptake of mannose derivatized GCD.

Animals, Materials and Experimental Procedures:

Mice:

BALB/C female mice 7-8 weeks, n=5.

i.v. Administration:

Mice are i.v. injected with derivatized GCD and untreated GCD. Animalsare sacrificed after 1, 2, 4 and 18 hours post-injection. The liver andspleen from each animal are removed, frozen in liquid nitrogen andstored at −70° C. until analysis.

Preparation of Liver and Spleen Tissue Samples:

Each tissue sample is washed with 0.9% NaCl and homogenized withhomogenization buffer (60 mM phosphate citrate, 1.5% Triton X-100, 1 mMPMSF), 5 ml buffer per gram tissue using a ULTRA-TURRAX T 25 basicIKA-WERKE homogenizer at low speed (11,000-13,000 l/min) for 45-60seconds, on ice. Samples are centrifuged at 10,000 g for 10 minutes at4° C. The supernatant is collected and divided to aliquots, and frozenat −70° C. until analysis.

In Vitro Glycosidase Activity Assay:

The activity of GCD taken up by the cells is determined by enzymaticactivity assay using p-nitrophenyl-beta-D-glucopyranoside. The assaybuffer contains 4 mM p-nitrophenyl-beta-D-glucopyranoside, 1.3 mM EDTA,0.15% Triton X-100, 0.125% sodium taurocholate, 60 mM phosphate-citratebuffer, pH 6.0. After 60 minutes at 37° C., the reaction is terminatedusing 5N NaOH, and the reaction product (p-nitrophenol) is determined byits absorbance at 405 nm.

Example 18 In-Vitro Uptake of Derivatized GFP

In order to investigate the general utility of M6P derivatization toincrease uptake into cells, GFP (green fluorescent protein) wasconjugated to an M6P-PEG₈-CH₂CH₂CO₂H moiety, using the proteinglycosylation procedure described hereinabove (see, Example 6.

Following 1 day of culture at 37° C., the cell medium was replaced withfresh serum-free medium containing 1 μM of GFP or 1 μM M6P-PEG-GFP.Cells were incubated for 24 hours and then visualized using aninverse-fluorescent microscope.

As shown in FIGS. 27A-B, uptake of M6P-PEG₈-GFP was greater than uptakeof GFP.

Example 19 Optimization of Reaction Conditions for Conjugation

α-Galctosidase A was conjugated with 5 kDa mPEG-COOH that waspreactivated with EDC and sulfo-NHS. The effect of reaction conditionson the efficacy of conjugation was tested.

The α-galctosidase A was reacted with 100 equivalents of 5 kDa mPEGusing various concentrations of EDC and sulfo-NHS. The molar ratios ofthe reactants in each group are summarized in Table 2.

TABLE 2 Molar equivalents of reactants in experimental groups group 1 23 4 5 6 7 8 PEG 100 100 100 100 100 100 100 100 EDC 100 130 200 100 100130 200 100 S-NHS 100 130 200 100 100 130 200 100 α-Gal 1 1 1 1 1 1 1 1

The preactivation of PEG in all the experiments was performed in DMSO.The coupling reaction was performed at pH=7.4.

The resulting conjugates were examined using SDS-PAGE.

As shown in FIG. 28, higher ratios of EDC and sulfo-NHS to PEG andprotein resulted in higher molecular weights, indicating more effectiveconjugation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

What is claimed is:
 1. A compound comprising a saccharide moiety and anon-hydrophobic linker being attached thereto, said linker comprising apoly(alkylene glycol) chain of at least 18 atoms in length, saidpoly(alkylene glycol) comprising 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20 alkylene glycol units.
 2. The compound of claim 1,wherein said linker is attached to an anomeric carbon of said saccharidemoiety.
 3. The compound of claim 2, wherein said linker is attached tosaid anomeric carbon via a bond having α configuration.
 4. The compoundof claim 1, wherein said poly(alkylene glycol) comprises poly(ethyleneglycol) (PEG).
 5. The compound of claim 1, wherein said poly(alkyleneglycol) is from 24 to 36 atoms in length.
 6. The compound of claim 4,wherein said poly(ethylene glycol) comprises from 8 to 12 ethyleneglycol units.
 7. The compound of claim 1, wherein said linker is abranched linker comprising at least two linear chemical moieties whichare covalently linked to one another, each of said linear chemicalmoieties being attached to a saccharide moiety, such that said linker isattached to at least two saccharide moieties.
 8. The compound of claim7, wherein said branched linker comprises at least two linearpoly(alkylene glycol) moieties which are covalently linked to oneanother.
 9. The compound of claim 1, wherein said saccharide moiety is amonosaccharide, and said linker is a non-saccharide linker.
 10. Thecompound of claim 9, wherein said monosaccharide is selected from thegroup consisting of a sialic acid, a mannose and a M6P.
 11. The compoundof claim 1, wherein said saccharide moiety is a hexose.
 12. The compoundof claim 11, wherein said hexose is a D-hexose.
 13. The compound ofclaim 1, wherein said linker comprises a single reactive group.
 14. Thecompound of claim 13, wherein said reactive group is capable of reactingwith a protein to form a covalent bond between said linker and saidprotein.
 15. The compound of claim 13, wherein said reactive group isselected from the group consisting of an amine, a maleimide and acarboxylate.
 16. The compound of claim 1 having the formula:

wherein: n=8; and Z is selected from the group consisting of —CH₂CH₂CO₂Hand —CH₂CH₂—NHCOCH₂CH₂-maleimide.
 17. A compound selected from the groupconsisting of:


18. A compound comprising a monosaccharide moiety and a non-hydrophobiclinker being attached thereto, said monosaccharide being selected fromthe group consisting of sialic acid and M6P and said linker being anon-saccharide linker and comprising a poly(alkylene glycol) being atleast 18 atoms in length.
 19. The compound of claim 18, wherein saidlinker comprises a single reactive group.
 20. A conjugate formed byreacting the compound of claim 14 with a protein, to thereby form saidcovalent bond between said linker and said protein.